Portable sensor array system

ABSTRACT

A system for the rapid characterization of multi-analyte fluids, in one embodiment, includes a light source, a sensor array, and a detector. The sensor array is formed from a supporting member into which a plurality of cavities may be formed. A series of chemically sensitive particles are, in one embodiment positioned within the cavities. The particles may be configured to produce a signal when a receptor coupled to the particle interacts with the analyte. Using pattern recognition techniques, the analytes within a multi-analyte fluid may be characterized.

PRIORITY CLAIM

This application claims priority to U.S. Provisional Application No.60/179,369 entitled “METHOD AND SYSTEM FOR COLLECTING AND TRANSMITTINGCHEMICAL INFORMATION,” filed Jan. 31, 2000, U.S. Provisional ApplicationNo. 60/179,424 entitled “SYSTEM AND METHOD FOR THE ANALYSIS OF BODILYFLUIDS” filed Jan. 31, 2000, U.S. Provisional Application No. 60/179,294entitled “SYSTEM AND METHOD FOR IDENTIFYING NUCLEIC ACIDS IN A FLUIDSAMPLE,” filed Jan. 31, 2000, U.S. Provisional Application No.60/179,380 entitled “METHOD OF PREPARING A SENSOR ARRAY,” filed Jan. 31,2000, U.S. Provisional Application No. 60/179,292 entitled “SYSTEM FORTRANSFERRING FLUID SAMPLES THROUGH A SENSOR ARRAY,” filed Jan. 31, 2000and U.S. Provisional Application No. 60/179,293 entitled “PORTABLESENSOR ARRAY SYSTEM,” filed Jan. 31, 2000.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Research leading to this invention was federally supported, in part, bygrant No. 1R01GM57306-01 entitled “The Development of an ElectronicTongue” from the National Institute of Health and the U.S. Governmenthas certain rights to this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method and device for the detectionof analytes in a fluid. More particularly, the invention relates to thedevelopment of a sensor array system capable of discriminating mixturesof analytes, toxins, and/or bacteria in medical, food/beverage, andenvironmental solutions.

2. Brief Description of the Related Art

The development of smart sensors capable of discriminating differentanalytes, toxins, and bacteria has become increasingly important forclinical, environmental, health and safety, remote sensing, military,food/beverage and chemical processing applications. Although manysensors capable of high sensitivity and high selectivity detection havebeen fashioned for single analyte detection, only in a few selectedcases have array sensors been prepared which display solution phasemulti-analyte detection capabilities. The advantages of such arraysystems are their utility for the analysis of multiple analytes andtheir ability to be “trained” to respond to new stimuli. Such on siteadaptive analysis capabilities afforded by the array structures maketheir utilization promising for a variety of future applications. Arraybased sensors displaying the capacity to sense and identify complexvapors have been demonstrated recently using a number of distincttransduction schemes. For example, functional sensors based on SurfaceAcoustic Wave (SAW), tin oxide (SnO₂) sensors, conductive organicpolymers, and carbon black/polymer composites have been fashioned. Theuse of tin oxide sensors, for example, is described in U.S. Pat. No.5,654,497 to Hoffheins et al. These sensors display the capacity toidentify and discriminate between a variety of organic vapors by virtueof small site-to-site differences in response characteristics. Patternrecognition of the overall fingerprint response for the array serves asthe basis for an olfaction-like detection of the vapor phase analytespecies. Indeed, several commercial “electronic noses” have beendeveloped recently. Most of the well established sensing elements arebased on SnO₂ arrays which have been derivatized so as to yieldchemically distinct response properties. Arrays based on SAW crystalsyield extremely sensitive responses to vapor, however, engineeringchallenges have prevented the creation of large SAW arrays havingmultiple sensor sites. To our knowledge, the largest SAW device reportedto date possesses only 12 sensor elements. Additionally, limitedchemical diversity and the lack of understanding of the molecularfeatures of such systems makes their expansion into more complexanalysis difficult.

Other structures have been developed that are capable of identifying anddiscriminating volatile organic molecules. One structure involves aseries of conductive polymer layers deposited onto metal contactinglayers. When these sensors are exposed to volatile reagents, some of thevolatile reagents adsorb into the polymer layers, leading to smallchanges in the electrical resistance of these layers. It is the smalldifferences in the behavior of the various sites that allows for adiscrimination, identification, and quantification of the vapors. Thedetection process takes only a few seconds, and sensitivities ofpart-per-billion can be achieved with this relatively simple approach.This “electronic nose” system is described in U.S. Pat. No. 5,698,089 toLewis et al. which is incorporated herein by reference as if set forthherein.

Although the above described electronic nose provides an impressivecapability for monitoring volatile reagents, the system possesses anumber of undesirable characteristics that warrant the development ofalternative sensor array systems. For example, the electronic nose canbe used only for the identification of volatile reagents. For manyenvironmental, military, medical, and commercial applications, theidentification and quantification of analytes present in liquid orsolid-phase samples is necessary. Moreover, the electronic nose systemsare expensive (e.g., the Aromascan system costs about $50,000/unit) andbulky (≧1 ft³). Furthermore, the functional elements for the currentlyavailable electronic nose are composed of conductive polymer systemswhich possess little chemical selectivity for many of the analytes whichare of interest to the military and civilian communities.

One of the most commonly employed sensing techniques has exploitedcolloidal polymer microspheres for latex agglutination tests (LATs) inclinical analysis. Commercially available LATs for more than 60 analytesare used routinely for the detection of infectious diseases, illegaldrugs, and early pregnancy tests. The vast majority of these types ofsensors operate on the principle of agglutination of latex particles(polymer microspheres) which occurs when the antibody-derivatizedmicrospheres become effectively “cross-linked” by a foreign antigenresulting in the attachment to, or the inability to pass through afilter. The dye-doped microspheres are then detected calorimetricallyupon removal of the antigen carrying solution.

However, the LATs lack the ability to be utilized for multiple, realtime analyte detection schemes as the nature of the responseintrinsically depends on a cooperative effect of the entire collectionof microspheres.

Similar to the electronic nose, array sensors that have shown greatanalytical promise are those based on the “DNA on a chip” technology.These devices possess a high density of DNA hybridization sites that areaffixed in a two-dimensional pattern on a planar substrate. To generatenucleotide sequence information, a pattern is created from unknown DNAfragments binding to various hybridization sites. Both radiochemical andoptical methods have provided excellent detection limits for analysis oflimited quantities of DNA. (Stimpson, D. I.; Hoijer, J. V.; Hsieh, W.;Jou, C.; Gardon, J.; Theriault, T.; Gamble, R.; Baldeschwieler, J. D.Proc. Natl. Acad. Sci. USA 1995, 92, 6379). Although quite promising forthe detection of DNA fragments, these arrays are generally not designedfor non-DNA molecules, and accordingly show very little sensitivity tosmaller organic molecules. Many of the target molecules of interest tocivilian and military communities, however, do not possess DNAcomponents. Thus, the need for a flexible, non-DNA based sensor is stilldesired. Moreover, while a number of prototype DNA chips containing upto a few thousand different nucleic acid probes have been described, theexisting technologies tend to be difficult to expand to a practicalsize. As a result, DNA chips may be prohibitively expensive forpractical uses.

Systems for analyzing fluid samples using an array formed ofheterogeneous, semi-selective thin films which function as sensingreceptor units are described in U.S. Pat. Nos. 6,023,540; 5,814,524;5,700,897; 5,512,490; 5,480,723; 5,252,494; 5,250,264; 5,244,813;5,244,636; and 5,143,853 which are incorporated herein by reference asif set forth herein. These systems appears to describe the use ofcovalently attached polymeric “cones” which are grown viaphotopolymerization onto the distal face of fiber optic bundles. Thesesensor probes appear to be designed with the goal of obtaining unique,continuous, and reproducible responses from small localized regions ofdye-doped polymer. The polymer appears to serve as a solid support forindicator molecules that provide information about test solutionsthrough changes in optical properties. These polymer supported sensorshave been used for the detection of analytes such as pH, metals, andspecific biological entities. Methods for manufacturing large numbers ofreproducible sensors, however, has yet to be developed. Moreover, nomethods for acquisitions of data streams in a simultaneous manner arecommercially available with this system. Optical alignment issues mayalso be problematic for these systems.

A method of rapid sample analysis for use in the diagnostic microbiologyfield is also desirable. The techniques now used for rapid microbiologydiagnostics detect either antigens or nucleic acids. Rapid antigentesting is based on the use of antibodies to recognize either the singlecell organism or the presence of infected cell material. Inherent tothis approach is the need to obtain and characterize the binding of theantibody to unique structures on the organism being tested. Since theidentification and isolation of the appropriate antibodies is timeconsuming, these techniques are limited to a single agent per testingmodule and there is no opportunity to evaluate the amount of agentpresent.

Most antibody methods are relatively insensitive and require thepresence of 10⁵ to 10⁷ organisms. The response time of antibody-antigenreactions in diagnostic tests of this type ranges from 10 to 120minutes, depending on the method of detection. The fastest methods aregenerally agglutination reactions, but these methods are less sensitivedue to difficulties in visual interpretation of the reactions.Approaches with slower reaction times include antigen recognition byantibody conjugated to either an enzyme or chromophore. These test typestend to be more sensitive, especially when spectrophotometric methodsare used to determine if an antigen-antibody reaction has occurred.These detection schemes do not, however, appear to allow thesimultaneous detection of multiple analytes on a single detectorplatform.

The alternative to antigen detection is the detection of nucleic acids.An approach for diagnostic testing with nucleic acids uses hybridizationto target unique regions of the target organism. These techniquesrequire fewer organisms (10³ to 10⁵), but require about five hours tocomplete. As with antibody-antigen reactions this approach has not beendeveloped for the simultaneous detection of multiple analytes.

The most recent improvement in the detection of microorganisms has beenthe use of nucleic acid amplification. Nucleic acid amplification testshave been developed that generate both qualitative and quantitativedata. However, the current limitations of these testing methods arerelated to delays caused by specimen preparation, amplification, anddetection. Currently, the standard assays require about five hours tocomplete. The ability to complete much faster detection for a variety ofmicroorganisms would be of tremendous importance to militaryintelligence, national safety, medical, environmental, and food areas.

It is therefore desirable that new sensors capable of discriminatingdifferent analytes, toxins, and bacteria be developed formedical/clinical diagnostic, environmental, health and safety, remotesensing, military, food/beverage, and chemical processing applications.It is further desired that the sensing system be adaptable to thesimultaneous detection of a variety of analytes to improve throughputduring various chemical and biological analytical procedures.

SUMMARY OF THE INVENTION

Herein we describe a system and method for the analysis of a fluidcontaining one or more analytes. The system may be used for eitherliquid or gaseous fluids. The system, in some embodiments, may generatepatterns that are diagnostic for both the individual analytes andmixtures of the analytes. The system in some embodiments, is made of aplurality of chemically sensitive particles, formed in an ordered array,capable of simultaneously detecting many different kinds of analytesrapidly. An aspect of the system is that the array may be formed using amicrofabrication process, thus allowing the system to be manufactured inan inexpensive manner.

In an embodiment of a system for detecting analytes, the system, in someembodiments, includes a light source, a sensor array, and a detector.The sensor array, in some embodiments, is formed of a supporting memberwhich is configured to hold a variety of chemically sensitive particles(herein referred to as “particles”) in an ordered array. The particlesare, in some embodiments, elements which will create a detectable signalin the presence of an analyte. The particles may produce optical (e.g.,absorbance or reflectance) or fluorescence/phosphorescent signals uponexposure to an analyte. Examples of particles include, but are notlimited to functionalized polymeric beads, agarous beads, dextrosebeads, polyacrylamide beads, control pore glass beads, metal oxidesparticles (e.g., silicon dioxide (SiO₂) or aluminum oxides (Al₂O₃)),polymer thin films, metal quantum particles (e.g., silver, gold,platinum, etc.), and semiconductor quantum particles (e.g., Si, Ge,GaAs, etc.). A detector (e.g., a charge-coupled device “CCD”) in oneembodiment is positioned below the sensor array to allow for the dataacquisition. In another embodiment, the detector may be positioned abovethe sensor array to allow for data acquisition from reflectance of thelight off of the particles.

Light originating from the light source may pass through the sensorarray and out through the bottom side of the sensor array. Lightmodulated by the particles may pass through the sensor array and ontothe proximally spaced detector. Evaluation of the optical changes may becompleted by visual inspection or by use of a CCD detector by itself orin combination with an optical microscope. A microprocessor may becoupled to the CCD detector or the microscope. A fluid delivery systemmay be coupled to the supporting member of the sensor array. The fluiddelivery system, in some embodiments, is configured to introduce samplesinto and out of the sensor array.

In an embodiment, the sensor array system includes an array ofparticles. The particles may include a receptor molecule coupled to apolymeric bead. The receptors, in some embodiments, are chosen forinteracting with analytes. This interaction may take the form of abinding/association of the receptors with the analytes. The supportingmember may be made of any material capable of supporting the particles,while allowing the passage of the appropriate wavelengths of light. Thesupporting member may include a plurality of cavities. The cavities maybe formed such that at least one particle is substantially containedwithin the cavity. In an embodiment, the optical detector may beintegrated within the bottom of the supporting member, rather than usinga separate detecting device. The optical detectors may be coupled to amicroprocessor to allow evaluation of fluids without the use of separatedetecting components. Additionally, a fluid delivery system may also beincorporated into the supporting member. Integration of detectors and afluid delivery system into the supporting member may allow the formationof a compact and portable analyte sensing system.

A high sensitivity CCD array may be used to measure changes in opticalcharacteristics which occur upon binding of the biological/chemicalagents. The CCD arrays may be interfaced with filters, light sources,fluid delivery and micromachined particle receptacles, so as to create afunctional sensor array. Data acquisition and handling may be performedwith existing CCD technology. CCD detectors may be configured to measurewhite light, ultraviolet light or fluorescence. Other detectors such asphotomultiplier tubes, charge induction devices, photo diodes,photodiode arrays, and microchannel plates may also be used.

A particle, in some embodiments, possess both the ability to bind theanalyte of interest and to create a modulated signal. The particle mayinclude receptor molecules which posses the ability to bind the analyteof interest and to create a modulated signal. Alternatively, theparticle may include receptor molecules and indicators. The receptormolecule may posses the ability to bind to an analyte of interest. Uponbinding the analyte of interest, the receptor molecule may cause theindicator molecule to produce the modulated signal. The receptormolecules may be naturally occurring or synthetic receptors formed byrational design or combinatorial methods.

Some examples of natural receptors include, but are not limited to, DNA,RNA, proteins, enzymes, oligopeptides, antigens, and antibodies. Eithernatural or synthetic receptors may be chosen for their ability to bindto the analyte molecules in a specific manner.

In one embodiment, a naturally occurring or synthetic receptor is boundto a polymeric bead in order to create the particle. The particle, insome embodiments, is capable of both binding the analyte(s) of interestand creating a detectable signal. In some embodiments, the particle willcreate an optical signal when bound to an analyte of interest.

A variety of natural and synthetic receptors may be used. The syntheticreceptors may come from a variety of classes including, but not limitedto, polynucleotides (e.g., aptamers), peptides (e.g., enzymes andantibodies), synthetic receptors, polymeric unnatural biopolymers (e.g.,polythioureas, polyguanidiniums), and imprinted polymers.Polynucleotides are relatively small fragments of DNA which may bederived by sequentially building the DNA sequence. Peptides includenatural peptides such as antibodies or enzymes or may be synthesizedfrom amino acids. Unnatural biopolymers are chemical structure which arebased on natural biopolymers, but which are built from unnatural linkingunits. For example, polythioureas and polyguanidiniums have a structuresimilar to peptides, but may be synthesized from diamines (i.e.,compounds which include at least two amine functional groups) ratherthan amino acids. Synthetic receptors are designed organic or inorganicstructures capable of binding various analytes.

In an embodiment, a large number of chemical/biological agents ofinterest to the military and civilian communities may be sensed readilyby the described array sensors. Bacteria may also be detected using asimilar system. To detect, sense, and identify intact bacteria, the cellsurface of one bacteria may be differentiated from other bacteria, orgenomic material may be detected using oligonucleic receptors. Onemethod of accomplishing this differentiation is to target cell surfaceoligosaccharides (i.e., sugar residues). The use of synthetic receptorswhich are specific for oligosaccharides may be used to determine thepresence of specific bacteria by analyzing for cell surfaceoligosaccharides.

In one embodiment, a receptor may be coupled to a polymeric resin. Thereceptor may undergo a chemical reaction in the presence of an analytesuch that a signal is produced. Indicators may be coupled to thereceptor or the polymeric bead. The chemical reaction of the analytewith the receptor may cause a change in the local microenvironment ofthe indicator to alter the spectroscopic properties of the indicator.This signal may be produced using a variety of signalling protocols.Such protocols may include absorbance, fluorescence resonance energytransfer, and/or fluorescence quenching. Receptor-analyte combinationmay include, but are not limited to, peptides-proteases,polynucleotides-nucleases, and oligosaccharides-oligosaccharide cleavingagents.

In one embodiment, a receptor and an indicator may be coupled to apolymeric resin. The receptor may undergo a conformational change in thepresence of an analyte such that a change in the local microenvironmentof the indicator occurs. This change may alter the spectroscopicproperties of the indicator. The interaction of the receptor with theindicator may be produce a variety of different signals depending on thesignalling protocol used. Such protocols may include absorbance,fluorescence resonance energy transfer, and/or fluorescence quenching.

In an embodiment, the sensor array system includes an array ofparticles. The particles may include a receptor molecule coupled to apolymeric bead. The receptors, in some embodiments, are chosen forinteracting with analytes. This interaction may take the form of abinding/association of the receptors with the analytes. The supportingmember may be made of any material capable of supporting the particles,while allowing the passage of the appropriate wavelengths of light. Thesupporting member may include a plurality of cavities. The cavities maybe formed such that at least one particle is substantially containedwithin the cavity. A vacuum may be coupled to the cavities. The vacuummay be applied to the entire sensor array. Alternatively, a vacuumapparatus may be coupled to the cavities to provide a vacuum to thecavities. A vacuum apparatus is any device capable of creating apressure differential to cause fluid movement. The vacuum apparatus mayapply a pulling force to any fluids within the cavity. The vacuumapparatus may pull the fluid through the cavity. Examples of vacuumapparatuss include pre-sealed vacuum chamber, vacuum pumps, vacuumlines, or aspirator-type pumps.

BRIEF DESCRIPTION OF THE DRAWINGS

The above brief description as well as further objects, features andadvantages of the methods and apparatus of the present invention will bemore fully appreciated by reference to the following detaileddescription of presently preferred but nonetheless illustrativeembodiments in accordance with the present invention when taken inconjunction with the accompanying drawings in which:

FIG. 1 depicts a schematic of an analyte detection system;

FIG. 2 depicts a particle disposed in a cavity;

FIG. 3 depicts a sensor array;

FIGS. 4A-F depict the formation of a Fabry-Perot cavity on the back of asensor array;

FIG. 5 depicts the chemical constituents of a particle;

FIG. 6 depicts the chemical formulas of some receptor compounds;

FIG. 7 depicts a plot of the absorbance of green light vs. concentrationof calcium. (Ca²⁺) for a particle which includes an o-cresolphthaleincomplexone receptor;

FIG. 8 depicts a schematic view of the transfer of energy from a firstindicator to a second indicator in the presence of an analyte;

FIG. 9 depicts a schematic of the interaction of a sugar molecule with aboronic acid based receptor.

FIG. 10 depicts various synthetic receptors;

FIG. 11 depicts a synthetic pathway for the synthesis of polythioureas;

FIG. 12 depicts a synthetic pathway for the synthesis ofpolyguanidiniums;

FIG. 13 depicts a synthetic pathway for the synthesis of diamines fromamino acids;

FIG. 14 depicts fluorescent diamino monomers;

FIG. 15 depicts a plot of counts/sec. (i.e., intensity) vs. time as thepH of a solution surrounding a particle coupled to o-cresolphthalein iscycled from acidic to basic conditions;

FIG. 16 depicts the color responses of a variety of sensing particles tosolutions of Ca⁺² and various pH levels;

FIG. 17 depicts an analyte detection system which includes a sensorarray disposed within a chamber;

FIG. 18 depicts an integrated analyte detection system;

FIG. 19 depicts a cross-sectional view of a cavity covered by a meshcover;

FIG. 20 depicts a top view of a cavity covered by a mesh cover;

FIGS. 21A-G depict a cross-sectional view of a series of processingsteps for the formation of a sensor array which includes a removable topand bottom cover;

FIGS. 22A-G depict a cross-sectional view of a series of processingsteps for the formation of a sensor array which includes a removable topand a stationary bottom cover;

FIGS. 23A-G depict a cross-sectional view of a series of processingsteps for the formation of a sensor array which includes a removabletop;

FIGS. 24A-D depict a cross-sectional view of a series of processingsteps for the formation of a silicon based sensor array which includes atop and bottom cover with openings aligned with the cavity;

FIGS. 25A-D depict a cross-sectional view of a series of processingsteps for the formation of a photoresist based sensor array whichincludes a top and bottom cover with openings aligned with the cavity;

FIGS. 26A-E depict a cross-sectional view of a series of processingsteps for the formation of a plastic based sensor array which includes atop and bottom cover with openings aligned with the cavity;

FIGS. 27A-D depict a cross-sectional view of a series of processingsteps for the formation of a silicon based sensor array which includes atop cover with openings aligned with the cavity and a tapered cavity;

FIGS. 28A-E depict a cross-sectional view of a series of processingsteps for the formation of a photoresist based sensor array whichincludes a top cover with openings aligned with the cavity and a taperedcavity;

FIGS. 29A-E depict a cross-sectional view of a series of processingsteps for the formation of a photoresist based sensor array whichincludes a top cover with openings aligned with the cavity and a bottomcover;

FIGS. 30A-D depict a cross-sectional view of a series of processingsteps for the formation of a plastic based sensor array which includes atop cover with openings aligned with the cavity and a bottom cover;

FIG. 31 depicts a cross-sectional view of a schematic of a micropump;

FIG. 32 depicts a top view of an electrohydrodynamic pump;

FIG. 33 depicts a cross-sectional view of a sensor array which includesa micropump;

FIG. 34 depicts a cross-sectional view of a sensor array which includesa micropump and channels which are coupled to the cavities;

FIG. 35 depicts a cross-sectional view of a sensor array which includesmultiple micropumps each micropump being coupled to a cavity;

FIG. 36 depicts a top view of a sensor array which includes multipleelectrohydrodynamic pumps;

FIG. 37 depicts a cross-sectional view of a sensor array which includesa system for delivering a reagent from a reagent particle to a sensingcavity;

FIG. 38 depicts a cross-sectional view of a sensor array which includesa vacuum chamber;

FIG. 39 depicts a cross-sectional view of a sensor array which includesa vacuum chamber, a filter, and a reagent reservoir.

FIG. 40 depicts a general scheme for the testing of an antibody analyte;

FIG. 41 depicts general scheme for the detection of antibodies whichuses a sensor array composed of four individual beads;

FIG. 42 depicts a sensor array which includes a vacuum chamber, a sensorarray chamber, and a sampling device;

FIG. 43 depicts a flow path of a fluid stream through a sensor arrayfrom the top toward the bottom of the sensor array;

FIG. 44 depicts a flow path of a fluid stream through a sensor arrayfrom the bottom toward the top of the sensor array;

FIGS. 45A-C depict the disruption of neuromuscular communication by atoxin;

FIG. 45D depicts the attachment of differentially protected lysine to abead;

FIG. 46 depicts a system for measuring the absorbance or emission of asensing particle;

FIG. 47 depicts receptors 3-6;

FIG. 48 depicts pH indicators which may be coupled to a particle;

FIG. 49 depicts a device for the analysis of IP₃ in cells;

FIG. 50 depicts the structure of Indo-1 and compound 2 and the emissionspectra of Indo-1 and compound 2 in the presence of Ca(II) and Ce(III),respectively;

FIG. 51 depicts a scheme wherein binding of citrate to a receptor freesup the Indo-1 for Ca(II) binding;

FIG. 52 depicts the change in FRET between coumarin and5-carboxyfluorescein on resin beads as a function of the solvent;

FIG. 53 depicts a scheme wherein a signal of apo-7 to citrate istriggered by Cu(II) binding;

FIG. 54 depicts the response of receptor 3 and 5-carboxyfluoroscein on aresin bead to the addition of citrate;

FIGS. 55A-I depict various sensing protocols forreceptor-indicator-polymeric resin particles;

FIG. 56 depicts a peptide trimer receptor and a pair of fluorescentindicators coupled to a polymeric resin;

FIG. 57 depicts a synthetic scheme for anchoring dansyl and dapoxylindicators to 6% agarose glyoxalated resin beads;

FIG. 58 depicts the RGB epifluorescence of 6 in EtOH with varying ratiobuffer concentrations;

FIG. 59 depicts indicators and polymeric beads used for fluorescencestudies;

FIG. 60 depicts Emission spectra of derivatized dapoxyl dyes in varioussolvents;

FIG. 61 depicts a general structure of a chemically sensitive particlethat includes a receptor and multiple indicators coupled to a polymericresin;

FIGS. 62A-D depict various sensing protocols forreceptor-indicator-polymeric resin particles in which a cleavagereaction occurs;

FIG. 63 depicts a plot of the fluorescence signal of a chemicallysensitive particle in the presence of trypsin;

FIG. 64 depicts a block diagram illustrating a system for collecting andtransmitting chemical information over a computer network;

FIG. 65 depicts a flowchart of a method for collecting and transmittingchemical information over a computer network;

FIG. 66 depicts a block diagram illustrating a system for collecting andtransmitting chemical information over a computer network;

FIG. 67 depicts a flowchart of a method for collecting and transmittingchemical information over a computer network;

FIG. 68 depicts a block diagram illustrating a system for collecting andtransmitting chemical information over a computer network;

FIG. 69 depicts a flowchart of a method for collecting and transmittingchemical information over a computer network;

FIGS. 70A-B depict a method of inserting particles into a sensor arrayusing a vacuum pickup dispenser head;

FIGS. 71A-B depict a method of inserting particles into a sensor arrayusing a solid dispenser head;

FIGS. 72A-D depict a method of inserting particles into a sensor arrayusing a vacuum chuck;

FIG. 73 depicts a cross section view of a sensor array which includes apassive pump system;

FIG. 74A depicts a top view of the sensor array of FIG. 57;

FIG. 74B depicts a bottom view of the sensor array of FIG. 57;

FIGS. 75A-D depict top views of the individual layers used to form asensor array;

FIG. 76 depicts a top view of a sensor array which includes multiplesuites of arrays;

FIG. 77 depicts an alternate cross sectional view of a sensor arraywhich includes a passive transport system;

FIG. 78 depicts a portable sensor array system;

FIG. 79A-B depict views of an alternate portable sensor array;

FIG. 80 depicts an exploded view of a cartridge for use in a portablesensor array;

FIG. 81 depicts a cross sectional view of a cartridge for use in aportable sensor array; and

FIG. 82 depicts the placement of a particle into a cavity.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Herein we describe a system and method for the simultaneous analysis ofa fluid containing multiple analytes. The system may be used for eitherliquid or gaseous fluids. The system may generate patterns that arediagnostic for both individual analytes and mixtures of the analytes.The system, in some embodiments, is made of a combination of chemicallysensitive particles, formed in an ordered array, capable ofsimultaneously detecting many different kinds of analytes rapidly. Anaspect of the system is that the array may be formed using amicrofabrication process, thus allowing the system to be manufactured inan inexpensive manner.

System for Analysis of Analytes

Shown in FIG. 1 is an embodiment of a system for detecting analytes in afluid. The system, in some embodiments, includes a light source 110, asensor array 120 and a detector 130. The light source 110 may be a whitelight source or light emitting diodes (LED). In one embodiment, lightsource 110 may be a blue light emitting diode (LED) for use in systemsrelying on changes in fluorescence signals. For colorimetric (e.g.,absorbance) based systems, a white light source may be used. The sensorarray 120, in some embodiments, is formed of a supporting member whichis configured to hold a variety of particles 124. A detecting device 130(e.g., a charge-coupled device “CCD”) may be positioned below the sensorarray to allow for data acquisition. In another embodiment, thedetecting device 130 may be positioned above the sensor array.

Light originating from the light source 110, in some embodiments, passesthrough the sensor array 120 and out through the bottom side of thesensor array. The supporting member and the particles together, in someembodiments, provide an assembly whose optical properties are wellmatched for spectral analyses. Thus, light modulated by the particlesmay pass through the sensor array and onto the proximally spaceddetector 130. Evaluation of the optical changes may be completed byvisual inspection (e.g., with a microscope) or by use of amicroprocessor 140 coupled to the detector. For fluorescencemeasurements, a filter 135 may be placed between supporting member 120and detector 130 to remove the excitation wavelength. A fluid deliverysystem 160 may be coupled to the supporting member. The fluid deliverysystem 160 may be configured to introduce samples into and out of thesensor array.

In an embodiment, the sensor array system includes an array ofparticles. Upon the surface and within the interior region of theparticles are, in some embodiments, located a variety of receptors forinteracting with analytes. The supporting member, in some embodiments,is used to localize these particles as well as to serve as amicroenvironment in which the chemical assays can be performed. For thechemical/biological agent sensor arrays, the particles used for analysisare about 0.05-500 microns in diameter, and may actually change size(e.g., swell or shrink) when the chemical environment changes.Typically, these changes occur when the array system is exposed to thefluid stream which includes the analytes. For example, a fluid streamwhich comprises a non-polar solvent, may cause non-polar particles tochange in volume when the particles are exposed to the solvent. Toaccommodate these changes, it is preferred that the supporting memberconsist of an array of cavities which serve as micro test-tubes.

The supporting member may be made of any material capable of supportingthe particles, while allowing the passage of the appropriate wavelengthof light. The supporting member is also made of a material substantiallyimpervious to the fluid in which the analyte is present. A variety ofmaterials may be used including plastics, glass, silicon based materials(e.g., silicon, silicon dioxide, silicon nitride, etc.) and metals. Inone embodiment, the supporting member includes a plurality of cavities.The cavities may be formed such that at least one particle issubstantially contained within the cavity. Alternatively, a plurality ofparticles may be contained within a single cavity.

In an embodiment, the supporting member may consist of a strip ofplastic which is substantially transparent to the wavelength of lightnecessary for detection. A series of cavities may be formed within thestrip. The cavities may be configured to hold at least one particle. Theparticles may be contained within the strip by a transparent cover whichis configured to allow passage of the analyte containing fluid into thecavities.

In another embodiment, the supporting member may be formed using asilicon wafer as depicted in FIG. 2. The silicon wafer 210 may include asubstantially transparent layer 220 formed on the bottom surface of thewafer. The cavities 230, in one embodiment, are formed by an anisotropicetch process of the silicon wafer. In one embodiment, anisotropicetching of the silicon wafer is accomplished using a wet hydroxide etch.Photolithographic techniques may be used to define the locations of thecavities. The cavities may be formed such that the sidewalls of thecavities are substantially tapered at an angle of between about 50 to 60degrees. Formation of such angled cavities may be accomplished by wetanisotropic etching of <100> silicon. The term “<100> silicon” refers tothe crystal orientation of the silicon wafer. Other types of silicon,(e.g., <110> and <111> silicon) may lead to steeper angled sidewalls.For example, <111> silicon may lead to sidewalls formed at about 90degrees. The angled sides of the cavities in some embodiments, serve as“mirror layers” which may improve the light collection efficiency of thecavities. The etch process may be controlled so that the formed cavitiesextend through the silicon wafer to the upper surface of transparentlayer 220. While depicted as pyramidal, the cavities may be formed in anumber of shapes including but not limited to, spherical, oval, cubic,or rectangular. An advantage to using a silicon wafer for the supportmember, is that the silicon material is substantially opaque to thelight produced from the light source. Thus, the light may be inhibitedfrom passing from one cavity to adjacent cavities. In this manner, lightfrom one cavity may be inhibited from influencing the spectroscopicchanges produced in an adjacent cavity.

The silicon wafer, in some embodiments, has an area of approximately 1cm² to about 100 cm² and includes about 10¹ to about 10⁶ cavities. In anembodiment, about 100 cavities are formed in a ten by ten matrix. Thecenter to center distance between the cavities, in some embodiments, isabout 500 microns. Each of the cavities may include at least oneparticle.

The transparent layer 220 may serve as a window, allowing light of avariety of wavelengths to pass through the cavities 230 and to thedetector. Additionally, the transparent layer may serve as a platformonto which the individual particles 235 may be positioned. Thetransparent layer may be formed of silicon dioxide (SiO₂), siliconnitride (Si₃N₄) or silicon dioxide/silicon nitride multi-layer stacks.The transparent layer, in some embodiments, is deposited onto thesilicon wafer prior to the formation of the cavities.

The cavities 230 may be sized to substantially contain a particle 235.The cavities are, in some embodiments, larger than a particle. Thecavities are, in some embodiments, sized to allow facile placement andremoval of the particle within the cavities. The cavity may besubstantially larger than the particle, thus allowing the particle toswell during use. For example, a particle may have a size as depicted inFIG. 2 by particle 235. During use, contact with a fluid (e.g., asolvent) may cause the particle to swell, for example, to a sizedepicted as circle 236. In some embodiments, the cavity is sized toallow such swelling of the particle during use. A particle may bepositioned at the bottom of a cavity using, e.g., a micromanipulator.After a particle has been placed within the cavity, a transparent coverplate 240 may be placed on top of the supporting member to keep theparticle in place.

When forming an array which includes a plurality of particles, theparticles may be placed in the array in an ordered fashion using themicromanipulator. In this manner, a ordered array having a predefinedconfiguration of particles may be formed. Alternatively, the particlesmay be randomly placed within the cavities. The array may subsequentlyundergo a calibration test to determine the identity of the particle atany specified location in the supporting member.

The transparent cover plate 240, in some embodiments, is coupled to theupper surface of the silicon wafer 220 such that the particles areinhibited from becoming dislodged from the cavity. The transparent coverplate, in some embodiments, is positioned a fixed distance above thesilicon wafer, as depicted in FIG. 2, to keep the particle in place,while allowing the entrance of fluids into the cavities. The transparentcover plate, in some embodiments, is positioned at a distance above thesubstrate which is substantially less than a width of the particle. Thetransparent cover plate may be made of any material which issubstantially transparent to the wavelength of light being utilized bythe detector. The transparent cover plate may be made of plastic, glass,quartz, or silicon dioxide/silicon nitride.

In one embodiment, the transparent cover plate 240, is a thin sheet ofglass (e.g., a microscope slide cover slip). The slide may be positioneda fixed distance above the silicon wafer. Support structures 241 (SeeFIG. 2) may be placed upon the silicon wafer 210 to position thetransparent cover plate 240. The support structures may be formed from apolymer or a silicon based material. In another embodiment, a polymericsubstrate is coupled to the silicon wafer to form the support structures241 for the transparent cover plate 240. In an embodiment, a plasticmaterial with an adhesive backing (e.g., cellophane tape) is positionedon the silicon wafer 210. After the support structures 241 are placed onthe wafer the transparent cover plate 240 is placed upon the supportstructures. The support structures inhibit the transparent cover sheetfrom contacting the silicon wafer 200. In this manner, a channel isformed between the silicon wafer and the transparent cover plate whichallow the fluid to pass into the cavity, while inhibiting displacementof the particle by the fluid.

In another embodiment, the transparent cover plate 240 may be fastenedto the upper surface of the silicon wafer, as depicted in FIG. 3. Inthis embodiment, the fluid may be inhibited from entering the cavities230 by the transparent cover plate 240. To allow passage of the fluidinto the cavities, a number of channels 250 may be formed in the siliconwafer. The channels, in one embodiment, are oriented to allow passage ofthe fluid into substantially all of the cavities. When contacted withthe fluid, the particles may swell to a size which may plug thechannels. To prevent this plugging, the channels may be formed near theupper portion of the cavities, as depicted in FIG. 3. The channels, inone embodiment, are formed using standard photolithographic masking todefine the regions where the trenches are to be formed, followed by theuse of standard etching techniques. A depth of the cavity may be suchthat the particle resides substantially below the position of thechannel. In this way, the plugging of the channels due to swelling ofthe particle may be prevented.

The inner surfaces of the cavities may be coated with a material to aidthe positioning of the particles within the cavities. In one embodiment,a thin layer of gold or silver may be used to line the inner surface ofthe cavities. The gold or silver layer may act as an anchoring surfaceto anchor particles (e.g., via alkylthiol bonding). In addition, thegold or silver layer may also increase the reflectivity of the innersurface of the cavities. The increased reflectance of the surface mayenhance the analyte detection sensitivity of the system. Alternatively,polymer layers and self-assembled monolayers formed upon the innersurface of the cavities may be used to control the particle adhesioninteractions. Additional chemical anchoring methods may be used forsilicon surfaces such as those based on siloxane type reagents, whichmay be attached to Si—OH functionalities. Similarly, monomeric andpolymeric reagents attached to an interior region of the cavities can beused to alter the local wetting characteristics of the cavities. Thistype of methodology can be used to anchor the particles as well as toalter the fluid delivery characteristics of the cavity. Furthermore,amplification of the signals for the analytes may be accomplished withthis type of strategy by causing preconcentration of appropriateanalytes in the appropriate type of chemical environment.

In another embodiment, the optical detector may be integrated within thebottom transparent layer 220 of the supporting member, rather than usinga separate detecting device. The optical detectors may be formed using asemiconductor-based photodetector 255. The optical detectors may becoupled to a microprocessor to allow evaluation of fluids without theuse of separate detecting components. Additionally, the fluid deliverysystem may also be incorporated into the supporting member. Micro-pumpsand micro-valves may also be incorporated into the silicon wafer to aidpassage of the fluid through the cavities. Integration of detectors anda fluid delivery system into the supporting member may allow theformation of a compact and portable analyte sensing system. Opticalfilters may also be integrated into the bottom membrane of the cavities.These filters may prevent short wavelength excitation from producing“false” signals in the optical detection system (e.g., a CCD detectorarray) during fluorescence measurements.

A sensing cavity may be formed on the bottom surface of the supportsubstrate. An example of a sensing cavity that may be used is aFabry-Perot type cavity. Fabry-Perot cavity-based sensors may be used todetect changes in optical path length induced by either a change in therefractive index or a change in physical length of the cavity. Usingmicromachining techniques, Fabry-Perot sensors may be formed on thebottom surface of the cavity.

FIGS. 4A-F depict a sequence of processing steps for the formation of acavity and a planar top diaphragm Fabry-Perot sensor on the bottomsurface of a silicon based supporting member. A sacrificial barrierlayer 262 a/b is deposited upon both sides of a silicon supportingmember 260. The silicon supporting member 260 may be a double-sidepolished silicon wafer having a thickness ranging from about 100 μm toabout 500 μm, preferably from about 200 μm to about 400 μm, and morepreferably of about 300 μm. The barrier layer 262 a/b may be composed ofsilicon dioxide, silicon nitride, or silicon oxynitride. In oneembodiment, the barrier layer 262 a/b is composed of a stack ofdielectric materials. As depicted in FIG. 4A, the barrier layer 262 a/bis composed of a stack of dielectric materials which includes a siliconnitride layer 271 a/b and a silicon dioxide layer 272 a/b. Both layersmay be deposited using a low pressure chemical vapor deposition(“LPCVD”) process. Silicon nitride may be deposited using an LPCVDreactor by reaction of ammonia (NH₃) and dichlorosilane (SiCl₂H₂) at agas flow rate of about 3.5:1, a temperature of about 800° C., and apressure of about 220 mTorr. The silicon nitride layer 271 a/b isdeposited to a thickness in the range from about 100 Å to about 500 Å,preferably from 200 Å to about 400 Å, and more preferably of about 300Å. Silicon dioxide is may be deposited using an LPCVD reactor byreaction of silane (SiH₄) and oxygen (O₂) at a gas flow rate of about3:4, a temperature of about 450° C., and a pressure of about 110 mTorr.The silicon dioxide layer 272 a/b is deposited to a thickness in therange from about 3000 Å to about 7000 Å, preferably from 4000 Å to about6000 Å, and more preferably of about 5000 Å. The front face silicondioxide layer 272 a, in one embodiment, acts as the main barrier layer.The underlying silicon nitride layer 271 a acts as an intermediatebarrier layer to inhibit overetching of the main barrier layer duringsubsequent KOH wet anisotropic etching steps.

A bottom diaphragm layer 264 a/b is deposited upon the barrier layer 262a/b on both sides of the supporting member 260. The bottom diaphragmlayer 264 a/b may be composed of silicon nitride, silicon dioxide, orsilicon oxynitride. In one embodiment, the bottom diaphragm layer 264a/b is composed of a stack of dielectric materials. As depicted in FIG.4A, the bottom diaphragm layer 264 a/b is composed of a stack ofdielectric materials which includes a pair of silicon nitride layers 273a/b and 275 a/b surrounding a silicon dioxide layer 274 a/b. All of thelayers may be deposited using an LPCVD process. The silicon nitridelayers 273 a/b and 275 a/b have a thickness in the range from about 500Å to about 1000 Å, preferably from 700 Å to about 800 Å, and morepreferably of about 750 Å. The silicon dioxide layer 274 a/b has athickness in the range from about 3000 Å to about 7000 Å, preferablyfrom 4000 Å to about 6000 Å, and more preferably of about 4500 Å.

A cavity which will hold the particle may now be formed in thesupporting member 260. The bottom diaphragm layer 264 b and the barrierlayer 262 b formed on the back side 261 of the silicon supporting member260 are patterned and etched using standard photolithographictechniques. In one embodiment, the layers are subjected to a plasma etchprocess. The plasma etching of silicon dioxide and silicon nitride maybe performed using a mixture of carbontetrafluoride (CF₄) and oxygen(O₂). The patterned back side layers 262 b and 264 b may be used as amask for anisotropic etching of the silicon supporting member 260. Thesilicon supporting member 260, in one embodiment, is anisotropicallyetched with a 40% potassium hydroxide (“KOH”) solution at 80° C. to formthe cavity. The etch is stopped when the front side silicon nitridelayer 271 a is reached, as depicted in FIG. 4B. The silicon nitridelayer 271 a inhibits etching of the main barrier layer 272 a during thisetch process. The cavity 267 may be formed extending through thesupporting member 260. After formation of the cavity, the remainingportions of the back side barrier layer 262 b and the diaphragm layer264 b may be removed.

Etch windows 266 are formed through the bottom diaphragm layer 264 a onthe front side of the wafer. A masking layer (not shown) is formed overthe bottom diaphragm layer 264 a and patterned using standardphotolithographic techniques. Using the masking layer, etch windows 266may be formed using a plasma etch. The plasma etching of silicon dioxideand silicon nitride may be performed using a mixture ofcarbontetrafluoride (CF₄) and oxygen (O₂). The etching is continuedthrough the bottom diaphragm layer 264 a and partially into the barrierlayer 262 a. In one embodiment, the etching is stopped at approximatelyhalf the thickness of the barrier layer 262 a. Thus, when the barrierlayer 262 a is subsequently removed the etch windows 266 will extendthrough the bottom diaphragm layer 264 a, communicating with the cavity267. By stopping the etching at a midpoint of the barrier layer, voidsor discontinuities may be reduced since the bottom diaphragm is stillcontinuous due to the remaining barrier layer.

After the etch windows 266 are formed, a sacrificial spacer layer 268a/b is deposited upon the bottom diaphragm layer 264 a and within cavity267, as depicted in FIG. 4C. The spacer layer may be formed from LPCVDpolysilicon. In one embodiment, the front side deposited spacer layer268 a will also at least partially fill the etch windows 266.Polysilicon may be deposited using an LPCVD reactor using silane (SiH₄)at a temperature of about 650° C. The spacer layer 268 a/b is depositedto a thickness in the range from about 4000 Å to about 10,000 Å,preferably from 6000 Å to about 8000 Å, and more preferably of about7000 Å. The preferred thickness of the spacer layer 268 a is dependenton the desired thickness of the internal air cavity of the Fabry-Perotdetector. For example, if a Fabry-Perot detector which is to include a7000 Åair cavity between the top and bottom diaphragm layer is desired,a spacer layer having a thickness of about 7000 Å would be formed. Afterthe spacer layer has been deposited, a masking layer for etching thespacer layer 268 a (not shown) is used to define the etch regions of thespacer layer 268 a. The etching may be performed using a composition ofnitric acid (HNO₃), water, and hydrogen fluoride (HF) in a ratio of25:13:1, respectively, by volume. The lateral size of the subsequentlyformed cavity is determined by the masking pattern used to define theetch regions of the spacer layer 268 a.

After the spacer layer 268 a has been etched, the top diaphragm layer270 a/b is formed. The top diaphragm 270 a/b, in one embodiment, isdeposited upon the spacer layer 268 a/b on both sides of the supportingmember. The top diaphragm 270 a/b may be composed of silicon nitride,silicon dioxide, or silicon oxynitride. In one embodiment, the topdiaphragm 270 a/b is composed of a stack of dielectric materials. Asdepicted in FIG. 4C, the top diaphragm 270 a/b is composed of a stack ofdielectric materials which includes a pair of silicon nitride layers 283a/b and 285 a/b surrounding a silicon dioxide layer 284 a/b. All of thelayers may be deposited using an LPCVD process. The silicon nitridelayers 283 a/b and 285 a/b have a thickness in the range from about 1000Å to about 2000 Å, preferably from 1200 Å to about 1700 Å, and morepreferably of about 1500 Å. The silicon dioxide layer 284 a/b has athickness in the range from about 5000 Å to about 15,500 Å, preferablyfrom 7500 Å to about 12,000 Å, and more preferably of about 10,500 Å.

After depositing the top diaphragm 270 a/b, all of the layers stacked onthe bottom face of the supporting member (e.g., layers 268 b, 283 b, 284b, and 285 b) are removed by multiple wet and plasma etching steps, asdepicted in FIG. 4D. After these layers are removed, the now exposedportions of the barrier layer 262 a are also removed. This exposes thespacer layer 268 a which is present in the etch windows 266. The spacerlayer 268 may be removed from between the top diaphragm 270 a and thebottom diaphragm 264 a by a wet etch using a KOH solution, as depictedin FIG. 4D. Removal of the spacer material 268 a, forms a cavity 286between the top diaphragm layer 270 a and the bottom diaphragm layer 264a. After removal of the spacer material, the cavity 286 may be washedusing deionized water, followed by isopropyl alcohol to clean out anyremaining etching solution.

The cavity 286 of the Fabry-Perot sensor may be filled with a sensingsubstrate 290, as depicted in FIG. 4E. To coat the cavity 286 with asensing substrate 290, the sensing substrate may be dissolved in asolvent. A solution of the sensing substrate is applied to thesupporting member 260. The solution is believed to rapidly enter thecavity 286 through the etched windows 266 in the bottom diaphragm 264 a,aided in part by capillary action. As the solvent evaporates, a thinfilm of the sensing substrate 290 coats the inner walls of the cavity286, as well as the outer surface of the bottom diaphragm 264 a. Byrepeated treatment of the supporting member with the solution of thesensing substrate, the thickness of the sensing substrate may be varied.

In one embodiment, the sensing substrate 290 is poly(3-dodecylthiophene)whose optical properties change in response to changes in oxidationstates. The sensing substrate poly(3-dodecylthiophene) may be dissolvedin a solvent such as chloroform or xylene. In one embodiment, aconcentration of about 0.1 g of poly(3-dodecylthiophene)/mL is used.Application of the solution of poly(3-dodecylthiophene) to thesupporting member causes a thin film of poly(3-dodecylthiophene) to beformed on the inner surface of the cavity.

In some instances, the sensing substrate, when deposited within a cavityof a Fabry-Perot type detector, may cause stress in the top diaphragm ofthe detector. It is believed that when a sensing polymer coats a planartop diaphragm, extra residual stress on the top diaphragm causes thediaphragm to become deflected toward the bottom diaphragm. If thedeflection becomes to severe, sticking between the top and bottomdiaphragms may occur. In one embodiment, this stress may be relieved bythe use of supporting members 292 formed within the cavity 286, asdepicted in FIG. 4F. The supporting members 292 may be formed withoutany extra processing steps to the above described process flow. Theformation of supporting members may be accomplished by deliberatelyleaving a portion of the spacer layer within the cavity. This may beaccomplished by underetching the spacer layer (e.g., terminating theetch process before the entire etch process is finished). The remainingspacer will behave as a support member to reduce the deflection of thetop diaphragm member. The size and shape of the support members may beadjusted by altering the etch time of the spacer layer, or adjusting theshape of the etch windows 266.

In another embodiment, a high sensitivity CCD array may be used tomeasure changes in optical characteristics which occur upon binding ofthe biological/chemical agents. The CCD arrays may be interfaced withfilters, light sources, fluid delivery and micromachined particlereceptacles, so as to create a functional sensor array. Data acquisitionand handling may be performed with existing CCD technology. Data streams(e.g., red, green, blue for colorimetric assays; gray intensity forfluorescence assays) may be transferred from the CCD to a computer via adata acquisition board. Current CCDs may allow for read-out rates of 10⁵pixels per second. Thus, the entire array of particles may be evaluatedhundreds of times per second allowing for studies of the dynamics of thevarious host-guest interaction rates as well as the analyte/polymerdiffusional characteristics. Evaluation of this data may offer a methodof identifying and quantifying the chemical/biological composition ofthe test samples. CCD detectors may be configured to measure whitelight, ultraviolet light or fluorescence. Other detectors such asphotomultiplier tubes, charge induction devices, photodiode, photodiodearrays, and microchannel plates may also be used. It should beunderstood that while the detector is depicted as being positioned underthe supporting member, the detector may also be positioned above thesupporting member. It should also be understood that the detectortypically includes a sensing element for detecting the spectroscopicevents and a component for displaying the detected events. The displaycomponent may be physically separated from the sensing element. Thesensing element may be positioned above or below the sensor array whilethe display component is positioned close to a user.

In one embodiment, a CCD detector may be used to record color changes ofthe chemical sensitive particles during analysis. As depicted in FIG. 1,a CCD detector 130 may be placed beneath the supporting member 120. Thelight transmitted through the cavities is captured and analyzed by theCCD detector. In one embodiment, the light is broken down into threecolor components, red, green and blue. To simplify the data, each coloris recorded using 8 bits of data. Thus, the data for each of the colorswill appear as a value between 0 and 255. The color of each chemicalsensitive element may be represented as a red, blue and green value. Forexample, a blank particle (i.e., a particle which does not include areceptor) will typically appear white. For example, when broken downinto the red, green and blue components, it is found that a typicalblank particle exhibits a red value of about 253, a green value of about250, and a blue value of about 222. This signifies that a blank particledoes not significantly absorb red, green or blue light. When a particlewith a receptor is scanned, the particle may exhibit a color change, dueto absorbance by the receptor. For example, it was found that when aparticle which includes a 5-carboxyfluorescein receptor is subjected towhite light, the particle shows a strong absorbance of blue light. TheCCD detector values for the 5-carboxyfluorescein particle exhibits a redvalue of about 254, a green value of about 218, and a blue value ofabout 57. The decrease in transmittance of blue light is believed to bedue to the absorbance of blue light by the 5-carboxyfluorescein. In thismanner, the color changes of a particle may be quantitativelycharacterized. An advantage of using a CCD detector to monitor the colorchanges is that color changes which may not be noticeable to the humaneye may now be detected.

The support array may be configured to allow a variety of detectionmodes to be practiced. In one embodiment, a light source is used togenerate light which is directed toward the particles. The particles mayabsorb a portion of the light as the light illuminates the particles.The light then reaches the detector, reduced in intensity by theabsorbance of the particles. The detector may be configure to measurethe reduction in light intensity (i.e., the absorbance) due to theparticles. In another embodiment, the detector may be placed above thesupporting member. The detector may be configured to measure the amountof light reflected off of the particles. The absorbance of light by theparticles is manifested by a reduction in the amount of light beingreflected from the cavity. The light source in either embodiment may bea white light source or a fluorescent light source.

Chemically Sensitive Particles

A particle, in some embodiments, possess both the ability to bind theanalyte of interest and to create a modulated signal. The particle mayinclude receptor molecules which posses the ability to bind the analyteof interest and to create a modulated signal. Alternatively, theparticle may include receptor molecules and indicators. The receptormolecule may posses the ability to bind to an analyte of interest. Uponbinding the analyte of interest, the receptor molecule may cause theindicator molecule to produce the modulated signal. The receptormolecules may be naturally occurring or synthetic receptors formed byrational design or combinatorial methods. Some examples of naturalreceptors include, but are not limited to, DNA, RNA, proteins, enzymes,oligopeptides, antigens, and antibodies. Either natural or syntheticreceptors may be chosen for their ability to bind to the analytemolecules in a specific manner. The forces which driveassociation/recognition between molecules include the hydrophobiceffect, anion-cation attraction, and hydrogen bonding. The relativestrengths of these forces depend upon factors such as the solventdielectric properties, the shape of the host molecule, and how itcomplements the guest. Upon host-guest association, attractiveinteractions occur and the molecules stick together. The most widelyused analogy for this chemical interaction is that of a “lock and key”.The fit of the key molecule (the guest) into the lock (the host) is amolecular recognition event.

A naturally occurring or synthetic receptor may be bound to a polymericresin in order to create the particle. The polymeric resin may be madefrom a variety of polymers including, but not limited to, agarous,dextrose, acrylamide, control pore glass beads, polystyrene-polyethyleneglycol resin, polystyrene-divinyl benzene resin, formylpolystyreneresin, trityl-polystyrene resin, acetyl polystyrene resin, chloroacetylpolystyrene resin, aminomethyl polystyrene-divinylbenzene resin,carboxypolystyrene resin, chloromethylated polystyrene-divinylbenzeneresin, hydroxymethyl polystyrene-divinylbenzene resin, 2-chlorotritylchloride polystyrene resin, 4-benzyloxy-2′4′-dimethoxybenzhydrol resin(Rink Acid resin), triphenyl methanol polystyrene resin,diphenylmethanol resin, benzhydrol resin, succinimidyl carbonate resin,p-nitrophenyl carbonate resin, imidazole carbonate resin, polyacrylamideresin, 4-sulfamylbenzoyl-4′-methylbenzhydrylamine-resin (Safety-catchresin), 2-amino-2-(2′-nitrophenyl) propionic acid-aminomethyl resin (ANPResin), p-benzyloxybenzyl alcohol-divinylbenzene resin (Wang resin),p-methylbenzhydrylamine-divinylbenzene resin (MBHA resin),Fmoc-2,4-dimethoxy-4′-(carboxymethyloxy)-benzhydrylamine linked to resin(Knorr resin), 4-(2′,4′-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin(Rink resin), 4-hydroxymethyl-benzoyl-4′-methylbenzhydrylamine resin(HMBA-MBHA Resin), p-nitrobenzophenone oxime resin (Kaiser oxime resin),and amino-2,4-dimethoxy-4′-(carboxymethyloxy)-benzhydrylamine handlelinked to 2-chlorotrityl resin (Knorr-2-chlorotrityl resin). In oneembodiment, the material used to form the polymeric resin is compatiblewith the solvent in which the analyte is dissolved. For example,polystyrene-divinyl benzene resin will swell within non-polar solvents,but does not significantly swell within polar solvents. Thus,polystyrene-divinyl benzene resin may be used for the analysis ofanalytes within non-polar solvents. Alternatively,polystyrene-polyethylene glycol resin will swell with polar solventssuch as water. Polystyrene-polyethylene glycol resin may be useful forthe analysis of aqueous fluids.

In one embodiment, a polystyrene-polyethylene glycol-divinyl benzenematerial is used to form the polymeric resin. Thepolystyrene-polyethylene glycol-divinyl benzene resin is formed from amixture of polystyrene 375, divinyl benzene 380 andpolystyrene-polyethylene glycol 385, see FIG. 5. The polyethylene glycolportion of the polystyrene-polyethylene glycol 385, in one embodiment,may be terminated with an amine. The amine serves as a chemical handleto anchor both receptors and indicator dyes. Other chemical functionalgroups may be positioned at the terminal end of the polyethylene glycolto allow appropriate coupling of the polymeric resin to the receptormolecules or indicators.

The chemically sensitive particle, in one embodiment, is capable of bothbinding the analyte(s) of interest and creating a detectable signal. Inone embodiment, the particle will create an optical signal when bound toan analyte of interest. The use of such a polymeric bound receptorsoffers advantages both in terms of cost and configurability. Instead ofhaving to synthesize or attach a receptor directly to a supportingmember, the polymeric bound receptors may be synthesized en masse anddistributed to multiple different supporting members. This allows thecost of the sensor array, a major hurdle to the development ofmass-produced environmental probes and medical diagnostics, to bereduced. Additionally, sensor arrays which incorporate polymeric boundreceptors may be reconfigured much more quickly than array systems inwhich the receptor is attached directly to the supporting member. Forexample, if a new variant of a pathogen or a pathogen that contains agenetically engineered protein is a threat, then a new sensor arraysystem may be readily created to detect these modified analytes bysimply adding new sensor elements (e.g., polymeric bound receptors) to apreviously formed supporting member.

In one embodiment, a receptor, which is sensitive to changes in the pHof a fluid sample is bound to a polymeric resin to create a particle.That is, the receptor is sensitive to the concentration of hydrogencations (H⁺). The receptor in this case is typically sensitive to theconcentration of H⁺ in a fluid solution. The analyte of interest maytherefore be H⁺. There are many types of molecules which undergo a colorchange when the pH of the fluid is changed. For example, many types ofdyes undergo significant color changes as the pH of the fluid medium isaltered. Examples of receptors which may be used to monitor the pH of afluid sample include 5-carboxyfluorescein and alizarin complexone,depicted in FIG. 6. Each of these receptors undergoes significant colorchanges as the pH of the fluid is altered. 5-carboxyfluoresceinundergoes a change from yellow to orange as the pH of the fluid isincreased. Alizarin complexone undergoes two color changes, first fromyellow to red, then from red to blue as the pH of the fluid increases.By monitoring the change in color caused by dyes attached to a polymericparticle, the pH of a solution may be qualitatively and, with the use ofa detector (e.g., a CCD detector), quantitatively monitored.

In another embodiment, a receptor which is sensitive to presence ofmetal cations is bound to a polymeric particle to create a particle. Thereceptor in this case is typically sensitive to the concentration of oneor more metal cations present in a fluid solution. In general, coloredmolecules which will bind cations may be used to determine the presenceof a metal cation in a fluid solution. Examples of receptors which maybe used to monitor the presence of cations in a fluid sample includealizarin complexone and o-cresolphthalein complexone, see FIG. 6. Eachof these receptors undergoes significant color changes as theconcentration of a specific metal ion in the fluid is altered. Alizarincomplexone is particularly sensitive to lanthanum ions. In the absenceof lanthanum, alizarin complexone will exhibit a yellow color. As theconcentration of lanthanum is increased, alizarin complexone will changeto a red color. o-Cresolphthalein complexone is particularly sensitiveto calcium ions. In the absence of calcium, o-cresolphthalein complexoneis colorless. As the concentration of calcium is increased,o-cresolphthalein complexone will change to a blue color. By monitoringthe change in color of metal cation sensitive receptors attached to apolymeric particle, the presence of a specific metal ion may bequalitatively and, with the use of a detector (e.g., a CCD detector),quantitatively monitored.

Referring to FIG. 7, a graph of the absorbance of green light vs.concentration of calcium (Ca⁺²) is depicted for a particle whichincludes an o-cresolphthalein complexone receptor. As the concentrationof calcium is increased, the absorbance of green light increases in alinear manner up to a concentration of about 0.0006 M. A concentrationof 0.0006 M is the solubility limit of calcium in the fluid, thus nosignificant change in absorbance is noted after this point. The linearrelationship between concentration and absorbance allows theconcentration of calcium to be determined by measuring the absorbance ofthe fluid sample.

In one embodiment, a detectable signal may be caused by the altering ofthe physical properties of an indicator ligand bound to the receptor orthe polymeric resin. In one embodiment, two different indicators areattached to a receptor or the polymeric resin. When an analyte iscaptured by the receptor, the physical distance between the twoindicators may be altered such that a change in the spectroscopicproperties of the indicators is produced. A variety of fluorescent andphosphorescent indicators may be used for this sensing scheme. Thisprocess, known as Forster energy transfer, is extremely sensitive tosmall changes in the distance between the indicator molecules.

For example, a first fluorescent indicator 320 (e.g., a fluoresceinderivative) and a second fluorescent indictor 330 (e.g., a rhodaminederivative) may be attached to a receptor 300, as depicted in FIG. 8.When no analyte is present short wavelength excitation 310 may excitethe first fluorescent indicator 320, which fluoresces as indicated by312. The short wavelength excitation, however, may cause little or nofluorescence of the second fluorescent indicator 330.

After binding of analyte 350 to the receptor, a structural change in thereceptor molecule may bring the first and second fluorescent indicatorscloser to each other. This change in intermolecular distance may allowthe excited first indicator 320 to transfer a portion of its fluorescentenergy 325 to the second fluorescent indicator 330. This transfer inenergy may be measured by either a drop in energy of the fluorescence ofthe first indicator molecule 320, or the detection of increasedfluorescence 314 by the second indicator molecule 330.

Alternatively, the first and second fluorescent indicators may initiallybe positioned such that short wavelength excitation, may causefluorescence of both the first and second fluorescent indicators, asdescribed above. After binding of analyte 350 to the receptor, astructural change in the receptor molecule may cause the first andsecond fluorescent indicators to move further apart. This change inintermolecular distance may inhibit the transfer of fluorescent energyfrom the first indicator 320 to the second fluorescent indicator 330.This change in the transfer of energy may be measured by either a dropin energy of the fluorescence of the second indicator molecule 330, orthe detection of increased fluorescence by the first indicator molecule320.

In another embodiment, an indicator ligand may be preloaded onto thereceptor. An analyte may then displace the indicator ligand to produce achange in the spectroscopic properties of the particles. In this case,the initial background absorbance is relatively large and decreases whenthe analyte is present. The indicator ligand, in one embodiment, has avariety of spectroscopic properties which may be measured. Thesespectroscopic properties include, but are not limited to, ultravioletabsorption, visible absorption, infrared absorption, fluorescence, andmagnetic resonance. In one embodiment, the indicator is a dye havingeither a strong fluorescence, a strong ultraviolet absorption, a strongvisible absorption, or a combination of these physical properties.Examples of indicators include, but are not limited to,carboxyfluorescein, ethidium bromide, 7-dimethylamino-4-methylcoumarin,7-diethylamino-4-methylcoumarin, eosin, erythrosin, fluorescein, OregonGreen 488, pyrene, Rhodamine Red, tetramethylrhodamine, Texas Red,Methyl Violet, Crystal Violet, Ethyl Violet, Malachite green, MethylGreen, Alizarin Red S, Methyl Red, Neutral Red,o-cresolsulfonephthalein, o-cresolphthalein, phenolphthalein, AcridineOrange, B-naphthol, coumarin, and a-naphthionic acid. When the indicatoris mixed with the receptor, the receptor and indicator interact witheach other such that the above mentioned spectroscopic properties of theindicator, as well as other spectroscopic properties may be altered. Thenature of this interaction may be a binding interaction, wherein theindicator and receptor are attracted to each other with a sufficientforce to allow the newly formed receptor-indicator complex to functionas a single unit. The binding of the indicator and receptor to eachother may take the form of a covalent bond, an ionic bond, a hydrogenbond, a van der Waals interaction, or a combination of these bonds.

The indicator may be chosen such that the binding strength of theindicator to the receptor is less than the binding strength of theanalyte to the receptor. Thus, in the presence of an analyte, thebinding of the indicator with the receptor may be disrupted, releasingthe indicator from the receptor. When released, the physical propertiesof the indicator may be altered from those it exhibited when bound tothe receptor. The indicator may revert back to its original structure,thus regaining its original physical properties. For example, if afluorescent indicator is attached to a particle that includes areceptor, the fluorescence of the particle may be strong beforetreatment with an analyte containing fluid. When the analyte interactswith the particle, the fluorescent indicator may be released. Release ofthe indicator may cause a decrease in the fluorescence of the particle,since the particle now has less indicator molecules associated with it.

An example of this type of system is illustrated by the use of a boronicacid substituted resin 505 as a particle. Prior to testing, the boronicacid substituted resin 505 is treated with a sugar 510 which is taggedwith an indicator (e.g., resorufin) as depicted in FIG. 9. The sugar 510binds to the boronic acid receptor 500 imparting a color change to theboronic substituted resin 505 (yellow for the resorufin tagged sugar).When the boronic acid resin 505 is treated with a fluid sample whichincludes a sugar 520, the tagged sugar 510 may be displaced, causing adecrease in the amount of color produced by the boronic acid substitutedresin 505. This decrease may be qualitatively or, with the use of adetector (e.g., a CCD detector), quantitatively monitored.

In another embodiment, a designed synthetic receptor may be used. In oneembodiment, a polycarboxylic acid receptor may be attached to apolymeric resin. The polycarboxylic receptors are discussed in U.S.patent application Ser. No. 08/950,712 which is incorporated herein byreference.

In an embodiment, the analyte molecules in the fluid may be pretreatedwith an indicator ligand. Pretreatment may involve covalent attachmentof an indicator ligand to the analyte molecule. After the indicator hasbeen attached to the analyte, the fluid may be passed over the sensingparticles. Interaction of the receptors on the sensing particles withthe analytes may remove the analytes from the solution. Since theanalytes include an indicator, the spectroscopic properties of theindicator may be passed onto the particle. By analyzing the physicalproperties of the sensing particles after passage of an analyte stream,the presence and concentration of an analyte may be determined.

For example, the analytes within a fluid may be derivatized with afluorescent tag before introducing the stream to the particles. Asanalyte molecules are adsorbed by the particles, the fluorescence of theparticles may increase. The presence of a fluorescent signal may be usedto determine the presence of a specific analyte. Additionally, thestrength of the fluorescence may be used to determine the amount ofanalyte within the stream.

Receptors

A variety of natural and synthetic receptors may be used. The syntheticreceptors may come from a variety of classes including, but not limitedto, polynucleotides (e.g., aptamers), peptides (e.g., enzymes andantibodies), synthetic receptors, polymeric unnatural biopolymers (e.g.,polythioureas, polyguanidiniums), and imprinted polymers, some of whichare generally depicted in FIG. 10. Natural based synthetic receptorsinclude receptors which are structurally similar to naturally occurringmolecules. Polynucleotides are relatively small fragments of DNA whichmay be derived by sequentially building the DNA sequence. Peptides maybe synthesized from amino acids. Unnatural biopolymers are chemicalstructure which are based on natural biopolymers, but which are builtfrom unnatural linking units. Unnatural biopolymers such aspolythioureas and polyguanidiniums may be synthesized from diamines(i.e., compounds which include at least two amine functional groups).These molecules are structurally similar to naturally occurringreceptors, (e.g., peptides). Some diamines may, in turn, be synthesizedfrom amino acids. The use of amino acids as the building blocks forthese compounds allow a wide variety of molecular recognition units tobe devised. For example, the twenty natural amino acids have side chainsthat possess hydrophobic residues, cationic and anionic residues, aswell as hydrogen bonding groups. These side chains may provide a goodchemical match to bind a large number of targets, from small moleculesto large oligosaccharides. Amino acid based peptides, polythioureas, andpolyguanidiniums are depicted in FIG. 10.

Techniques for the building of DNA fragments and polypeptide fragmentson a polymer particle are well known. Techniques for the immobilizationof naturally occurring antibodies and enzymes on a polymeric resin arealso well known. The synthesis of polythioureas upon a resin particlemay be accomplished by the synthetic pathway depicted in FIG. 11. Theprocedure may begin by deprotection of the terminal tBoc protectinggroup on an amino acid coupled to a polymeric particle. Removal of theprotecting group is followed by coupling of the rigid spacer 410 to theresulting amine 405 using diisopropylcarbodiimide (DIC) and1-hydroxybenzotriazole hydrate (HOBT). The spacer group may inhibitformation of a thiazolone by reaction of the first amino acids withsubsequently formed thioureas. After the spacer group is coupled to theamino acid, another tBoc deprotection is performed to remove the spacerprotecting group, giving the amine 415. At this point, monomer may beadded incrementally to the growing chain, each time followed by a tBocdeprotection. The addition of a derivative of the diamine 420 (e.g., anisothiocyanate) to amine 415 gives the mono-thiourea 425. The additionof a second thiourea substituent is also depicted. After the addition ofthe desired number of monomers, a solution of benzylisothiocyanate oracetic anhydride may be added to cap any remaining amines on the growingoligomers. Between 1 to 20 thioureas groups may be formed to produce asynthetic polythiourea receptor.

The synthesis of polyguanidiniums may be accomplished as depicted inFIG. 12. In order to incorporate these guanidinium groups into thereceptor, the coupling of a thiourea with a terminal amine in thepresence of Mukaiyama's reagent may be utilized. The coupling of thefirst thiourea diamine 430 with an amino group of a polymeric particlegives the mono-guanidinium 434. Coupling of the resultingmono-guanidinium with a second thiourea diamine 436 gives adi-guanidinium 438. Further coupling may create a tri-guanidinium 440.Between 1 to 20 guanidinium groups may be formed to produce a syntheticpolyguanidinium receptor.

The above described methods for making polythioureas andpolyguanidiniums are based on the incorporation of diamines (i.e.,molecules which include at least two amine functional groups) into theoligomeric receptor. The method may be general for any compound havingat least two amino groups. In one embodiment, the diamine may be derivedfrom amino acids. A method for forming diamines from amino acids isshown in FIG. 13. Treatment of a protected amino acid 450 withborane-THF reduces the carboxylic acid portion of the amino acid to theprimary alcohol 452. The primary alcohol is treated with phthalimideunder Mitsunobu conditions (PPh₃/DEAD). The resulting compound 454 istreated with aqueous methylamine to form the desired monoprotecteddiamine 456. The process may be accomplished such that the enantiomericpurity of the starting amino acid is maintained. Any natural orsynthetic amino acid may be used in the above described method.

The three coupling strategies used to form the respective functionalgroups may be completely compatible with each other. The capability tomix linking groups (amides, thioureas, and guanidiniums) as well as theside chains (hydrophobic, cationic, anionic, and hydrogen bonding) mayallow the creation of a diversity in the oligomers that is beyond thediversity of receptors typically found with natural biologicalreceptors. Thus, we may produce ultra-sensitive and ultra-selectivereceptors which exhibit interactions for specific toxins, bacteria, andenvironmental chemicals. Additionally, these synthetic schemes may beused to build combinatorial libraries of particles for use in the sensorarray. In an embodiment, the indicator ligand may be incorporated intosynthetic receptors during the synthesis of the receptors. The ligandmay be incorporated into a monomeric unit, such as a diamine, that isused during the synthesis of the receptor. In this manner, the indicatormay be covalently attached to the receptor in a controlled position. Byplacing the indicator within the receptor during the synthesis of thereceptor, the positioning of the indicator ligand within the receptormay be controlled. This control may be difficult to achieve aftersynthesis of the receptor is completed.

In one embodiment, a fluorescent group may be incorporated into adiamine monomer for use in the synthetic sequences. Examples ofmonomeric units which may be used for the synthesis of a receptor aredepicted in FIG. 14. The depicted monomers include fluorescent indicatorgroups. After synthesis, the interaction of the receptor with theanalyte may induce changes in the spectroscopic properties of themolecule. Typically, hydrogen bonding or ionic substituents on thefluorescent monomer involved in analyte binding have the capacity tochange the electron density and/or rigidity of the fluorescent ringsystem, thereby causing observable changes in the spectroscopicproperties of the indicator. For fluorescent indicators such changes maybe exhibited as changes in the fluorescence quantum yield, maximumexcitation wavelength, and/or maximum emission wavelength. This approachdoes not require the dissociation of a preloaded fluorescent ligand,which may be limited in response time by k_((off))). While fluorescentligands are shown here, it is to be understood that a variety of otherligand may be used including colorimetric ligands.

In another embodiment, two fluorescent monomers for signaling may beused for the synthesis of the receptor. For example, compound 470 (aderivative of fluorescein) and compound 475 (a derivative of rhodamine),depicted in FIG. 14, may both be incorporated into a synthetic receptor.Compound 470 contains a common colorimetric/fluorescent probe that will,in some embodiments, send out a modulated signal upon analyte binding.The modulation may be due to resonance energy transfer to compound 475.When an analyte binds to the receptor, structural changes in thereceptor may alter the distance between monomeric units 470 and 475. Itis well known that excitation of fluorescein can result in emission fromrhodamine when these molecules are oriented correctly. The efficiency ofresonance energy transfer from monomers 470 to 475 will depend stronglyupon the presence of analyte binding; thus, measurement of rhodaminefluorescence intensity (at a substantially longer wavelength thanfluorescein fluorescence) may serve as an indicator of analyte binding.To greatly improve the likelihood of a modulatory fluorescein-rhodamineinteraction, multiple rhodamine tags may be attached at different sitesalong a receptor molecule without substantially increasing backgroundrhodamine fluorescence (only rhodamine very close to fluorescein willyield appreciable signal). This methodology may be applied to a numberof alternate fluorescent pairs.

In an embodiment, a large number of chemical/biological agents ofinterest to the military and civilian communities may be sensed readilyby the described array sensors including both small and medium sizemolecules. For example, it is known that nerve gases typically producephosphate structures upon hydrolysis in water. The presence of moleculeswhich contain phosphate functional groups may be detected usingpolyguanidiniums. Nerve gases which have contaminated water sources maybe detected by the use of the polyguanidinium receptors described above.

In order to identify, sense, and quantitate the presence of variousbacteria using the proposed micro-machined sensor, two strategies may beused. First, small molecule recognition and detection may be exploited.Since each bacteria possesses a unique and distinctive concentration ofthe various cellular molecules, such as DNA, proteins, metabolites, andsugars, the fingerprint (i.e., the concentration and types of DNA,proteins, metabolites, and sugars) of each organism is expected to beunique. Hence, the analytes obtained from whole bacteria or broken downbacteria may be used to determine the presence of specific bacteria. Aseries of receptors specific for DNA molecules, proteins, metabolites,and sugars may be incorporated into an array. A solution containingbacteria, or more preferably broken down bacteria, may be passed overthe array of particles. The individual cellular components of thebacteria may interact in a different manner with each of the particles.This interaction will provide a pattern within the array which may beunique for the individual bacteria. In this manner, the presence ofbacteria within a fluid may be determined.

In another embodiment, bacteria may be detected as whole entities, asfound in ground water, aerosols, or blood. To detect, sense, andidentify intact bacteria, the cell surface of one bacteria may bedifferentiated from other bacteria. One method of accomplishing thisdifferentiation is to target cell surface oligosaccharides (i.e. sugarresidues). Each bacterial class (gram negative, gram positive, etc.)displays a different oligosaccharide on their cell surfaces.

The oligosaccharide, which is the code that is read by other cellsgiving an identification of the cell, is part of the cell-cellrecognition and communication process. The use of synthetic receptorswhich are specific for oligosaccharides may be used to determine thepresence of specific bacteria by analyzing for the cell surfaceoligosaccharides.

In another embodiment, the sensor array may be used to optimize whichreceptor molecules should be used for a specific analyte. An array ofreceptors may be placed within the cavities of the supporting member anda stream containing an analyte may be passed over the array. Thereaction of each portion of the sensing array to the known analyte maybe analyzed and the optimal receptor determined by determining whichparticle, and therefore which receptor, exhibits the strongest reactiontoward the analyte. In this manner, a large number of potentialreceptors may be rapidly scanned. The optimal receptor may then beincorporated into a system used for the detection of the specificanalyte in a mixture of analytes.

It should be emphasized that although some particles may be purposefullydesigned to bind to important species (biological agents, toxins, nervegasses, etc.), most structures will possess nonspecific receptor groups.One of the advantages associated with the proposed sensor array is thecapacity to standardize each array of particles via exposure to variousanalytes, followed by storage of the patterns which arise frominteraction of the analytes with the particles. Therefore, there may notbe a need to know the identity of the actual receptor on each particle.Only the characteristic pattern for each array of particles isimportant. In fact, for many applications it may be less time consumingto place the various particles into their respective holders withouttaking precautions to characterize the location associated with thespecific particles. When used in this manner, each individual sensorarray may require standardization for the type of analyte to be studied.

On-site calibration for new or unknown toxins may also be possible withthis type of array. Upon complexation of an analyte, the localmicroenvironment of each indicator may change, resulting in a modulationof the light absorption and/or emission properties. The use of standardpattern recognition algorithms completed on a computer platform mayserves as the intelligence factor for the analysis. The “fingerprint”like response evoked from the simultaneous interactions occurring atmultiple sites within the substrate may be used to identify the speciespresent in unknown samples.

The above described sensor array system offers a number of distinctadvantages over exiting technologies. One advantage is that “real time”detection of analytes may be performed. Another advantage is that thesimultaneous detection of multiple analytes may be realized. Yet anotheradvantage is that the sensor array system allows the use of syntheticreagents as well as biologically produced reagents. Synthetic reagentstypically have superior sensitivity and specificity toward analytes whencompared to the biological reagents. Yet another advantage is that thesensor array system may be readily modified by simply changing theparticles which are placed within the sensor array. Thisinterchangeability may also reduce production costs.

EXAMPLES 1. The Determination of pH Using a Chemically SensitiveParticle

Shown in FIG. 15 is the magnitude of the optical signal transmittedthrough a single polymer particle derivatized with o-cresolphthalein.Here, a filter is used to focus the analysis on those wavelengths whichthe dye absorbs most strongly (i.e., about 550 nm). Data is provided forthe particle as the pH is cycled between acid and basic environments. Inacidic media (i.e., at times of 100-150 seconds and 180-210 seconds),the particle is clear and the system yields large signals (up to greaterthan 300,000 counts) at the optical detector. Between times of 0-100 and150-180 seconds, the solution was made basic. Upon raising the pH (i.e.,making the solution more basic), the particle turns purple in color andthe transmitted green light is greatly diminished. Large signalreductions are recorded under such circumstances. The evolution of thesignal changes show that the response time is quite rapid, on the orderof 10 seconds. Furthermore, the behavior is highly reproducible.

2. The Simultaneous Detection of Ca⁺², Ce⁺³, and pH by a Sensor ArraySystem

The synthesis of four different particles was accomplished by coupling avariety of indictor ligands to a polyethylene glycol-polystyrene(“PEG-PS”) resin particle. The PEG-PS resin particles were obtained fromNovabiochem Corp., La Jolla, Calif. The particles have an averagediameter of about 130 μm when dry and about 250 μm when wet. Theindicator ligands of fluorescein, o-cresolphthalein complexone, andalizarin complexone were each attached to PEG-PS resin particles using adicyclohexylcarbodiimide (DCC) coupling between a terminal resin boundamine and a carboxylic acid on the indicator ligand.

These synthetic receptors, localized on the PEG-PS resin to createsensing particles, were positioned within micromachined wells formed insilicon/silicon nitride wafers, thus confining the particles toindividually addressable positions on a multicomponent chip. These wellswere sized to hold the particles in both swollen and unswollen states.Rapid introduction of the test fluids can be accomplished using thesestructures while allowing spectrophotometric assays to probe for thepresence of analytes. For the identification and quantification ofanalyte species, changes in the light absorption and light emissionproperties of the immobilized resin particles can be exploited, althoughonly identification based upon absorption properties are discussed here.Upon exposure to analytes, color changes for the particles were found tobe 90% complete within one minute of exposure, although typically onlyseconds were required. To make the analysis of the colorimetric changesefficient, rapid, and sensitive, a charge-coupled-device (CCD) wasdirectly interfaced with the sensor array. Thus, data streams composedof red, green, and blue (RGB) light intensities were acquired andprocessed for each of the individual particle elements. The red, blue,and green responses of the particles to various solutions aregraphically depicted in FIG. 16.

The true power of the described bead sensor array occurs whensimultaneous evaluation of multiple chemically distinct bead structuresis completed. A demonstration of the capacity of five different beads isprovided in FIG. 16. In this case, blank, alizarin, o-cresol phthalein,fluorescein, and alizarin-Ce3+ complex derivatized beads serve as amatrix for subtle differentiation of chemical environments. The blankbead is simply a polystyrene sphere with no chemical derivatization. Thebead derivatized with o-cresolphthalein responds to Ca+2 at pHs valuesaround 10.0. The binding of calcium is noted from the large green colorattenuation noted for this dye while exposed to the cation. Similarly,the fluorescein derivatized bead acts as a pH sensor. At pHs below 7.4it is light yellow, but at higher pHs it turns dark orange. Interesting,the alizarin complexone plays three distinct roles. First, it acts as aproton sensor yielding a yellow color at pHs below 4.5, orange is notedat pHs between 4.5 and 11.5, and at pHs above 11.5 a blue hue isobserved. Second, it functions as a sensor for lanthanum ions at lowerpHs by turning yellow to orange. Third, the combination of both fluorideand lanthanum ions results in yellow/orange coloration.

The analysis of solutions containing various amount of Ca⁺² or F⁻ atvarious pH levels was performed using alizarin complexone,o-cresolphthalein complexone, 5-carboxy fluorescein, and alizarin-Ce³⁺complex. A blank particle in which the terminal amines of a PEG-PS resinparticle have been acylated was also used. In this example, the presenceof Ca⁺² (0.1 M Ca(NO₃)₂) was analyzed under conditions of varying pH.The pH was varied to values of 2, 7, and 12, all buffered by a mixtureof 0.04 M phosphate, 0.04 M acetate, and 0.04 M borate. The RGB patternsfor each sensor element in all environments were measured. The beadderivatized with o-cresolphthalein responds to Ca⁺² at pH values around12. Similarly, the 5-carboxy fluorescein derivatized bead acts as a pHsensor. At pHs below 7.4 it is light yellow, but at higher pHs it turnsdark orange. Interesting, the alizarin complexone plays three distinctroles. First, it acts as a proton sensor yielding a yellow color at pHsbelow 4.5, orange is noted at pHs between 4.5 and 11.5, and at pHs above11.5 a blue hue is observed. Second, it functions as a sensor forlanthanum ions at lower pHs by turning yellow to orange. Third, thecombination of both fluoride and lanthanum ions results in yellow/orangecoloration.

This example demonstrates a number of important factors related to thedesign, testing, and functionality of micromachined array sensors forsolution analyses. First, derivatization of polymer particles with bothcolorimetric and fluorescent dyes was completed. These structures wereshown to respond to pH and Ca²⁺. Second, response times well under 1minute were found. Third, micromachined arrays suitable both forconfinement of particles, as well as optical characterization of theparticles, have been prepared. Fourth, integration of the test bedarrays with commercially available CCD detectors has been accomplished.Finally, simultaneous detection of several analytes in a mixture wasmade possible by analysis of the RGB color patterns created by thesensor array.

3. The Detection of Sugar Molecules Using a Boronic Acid Based Receptor

A series of receptors were prepared with functionalities that associatestrongly with sugar molecules, as depicted in FIG. 9. In this case, aboronic acid sugar receptor 500 was utilized to demonstrate thefunctionality of a new type of sensing scheme in which competitivedisplacement of a resorufin derivatized galactose sugar molecule wasused to assess the presence (or lack thereof) of other sugar molecules.The boronic acid receptor 500 was formed via a substitution reaction ofa benzylic bromide. The boronic acid receptor was attached to apolyethylene glycol-polystyrene (“PEG-PS”) resin particle at the “R”position. Initially, the boronic acid derivatized particle was loadedwith resorufin derivatized galactose 510. Upon exposure of the particleto a solution containing glucose 520, the resorufin derivatizedgalactose molecules 510 are displaced from the particle receptor sites.Visual inspection of the optical photographs taken before and afterexposure to the sugar solution show that the boron substituted resin iscapable of sequestering sugar molecules from an aqueous solution.Moreover, the subsequent exposure of the colored particles to a solutionof a non-tagged sugar (e.g., glucose) leads to a displacement of thebound colored sugar reporter molecule. Displacement of this moleculeleads to a change in the color of the particle. The sugar sensor turnsfrom dark orange to yellow in solutions containing glucose. Theparticles were also tested in conditions of varying pH. It was notedthat the color of the particles changes from dark orange to yellow asthe pH is varied from low pH to high pH.

Further Improvements 1. System Improvements

Shown in FIG. 17 is an embodiment of a system for detecting analytes ina fluid. In one embodiment, the system includes a light source 512, asensor array 522, a chamber 550 for supporting the sensor array and adetector 530. The sensor array 522 may include a supporting member whichis configured to hold a variety of particles. In one embodiment, lightoriginating from the light source 512 passes through the sensor array522 and out through the bottom side of the sensor array. Light modulatedby the particles may be detected by a proximally spaced detector 530.While depicted as being positioned below the sensor array, it should beunderstood that the detector may be positioned above the sensor arrayfor reflectance measurements. Evaluation of the optical changes may becompleted by visual inspection (e.g., by eye, or with the aid of amicroscope) or by use of a microprocessor 540 coupled to the detector.

In this embodiment, the sensor array 522 is positioned within a chamber550. The chamber 550, may be configured to allow a fluid stream to passthrough the chamber such that the fluid stream interacts with the sensorarray 522. The chamber may be constructed of glass (e.g, borosilicateglass or quartz) or a plastic material which is transparent to a portionof the light from the light source. If a plastic material is used, theplastic material should also be substantially unreactive toward thefluid. Examples of plastic materials which may be used to form thechamber include, but are not limited to, acrylic resins, polycarbonates,polyester resins, polyethylenes, polyimides, polyvinyl polymers (e.g.,polyvinyl chloride, polyvinyl acetate, polyvinyl dichloride, polyvinylfluoride, etc.), polystyrenes, polypropylenes, polytetrafluoroethylenes,and polyurethanes. An example of such a chamber is a Sykes-Moorechamber, which is commercially available from Bellco Glass, Inc., in NewJersey. Chamber 550, in one embodiment, includes a fluid inlet port 552and a fluid outlet port 554. The fluid inlet 552 and outlet 554 portsare configured to allow a fluid stream to pass into the interior 556 ofthe chamber during use. The inlet and outlet ports may be configured toallow facile placement of a conduit for transferring the fluid to thechamber. In one embodiment, the ports may be hollow conduits. The hollowconduits may be configured to have an outer diameter which issubstantially equal to the inner diameter of a tube for transferring thefluid to or away from the chamber. For example, if a plastic or rubbertube is used for the transfer of the fluid, the internal diameter of theplastic tube is substantially equal to the outer diameter of the inletand outlet ports.

In another embodiment, the inlet and outlet ports may be Luer lock styleconnectors. Preferably, the inlet and outlet ports are female Luer lockconnectors. The use of female Luer lock connectors will allow the fluidto be introduced via a syringe. Typically, syringes include a male Luerlock connector at the dispensing end of the syringe. For theintroduction of liquid samples, the use of Luer lock connectors mayallow samples to be transferred directly from a syringe to the chamber550. Luer lock connectors may also allow plastic or rubber tubing to beconnected to the chamber using Luer lock tubing connectors.

The chamber may be configured to allow the passage of a fluid sample tobe substantially confined to the interior 556 of the chamber. Byconfining the fluid to a small interior volume, the amount of fluidrequired for an analysis may be minimized. The interior volume may bespecifically modified for the desired application. For example, for theanalysis of small volumes of fluid samples, the chamber may be designedto have a small interior chamber, thus reducing the amount of fluidneeded to fill the chamber. For larger samples, a larger interiorchamber may be used. Larger chambers may allow a faster throughput ofthe fluid during use.

In another embodiment, depicted in FIG. 18, a system for detectinganalytes in a fluid includes a light source 512, a sensor array 522, achamber 550 for supporting the sensor array and a detector 530, allenclosed within a detection system enclosure 560. As described above,the sensor array 522 is preferably formed of a supporting member whichis configured to hold a variety of particles. Thus, in a singleenclosure, all of the components of an analyte detection system areincluded.

The formation of an analyte detection system in a single enclosure mayallow the formation of a portable detection system. For example, a smallcontroller 570 may be coupled to the analyte detection system. Thecontroller 570 may be configured to interact with the detector anddisplay the results from the analysis. In one embodiment, the controllerincludes a display device 572 for displaying information to a user. Thecontroller may also include input devices 574 (e.g., buttons) to allowthe user to control the operation of the analyte detection system. Forexample, the controller may control the operation of the light source512 and the operation of the detector 530.

The detection system enclosure 560, may be interchangeable with thecontroller. Coupling members 576 and 578 may be used to remove thedetection system enclosure 560 from the controller 570. A seconddetection system enclosure may be readily coupled to the controllerusing coupling members 576 and 578. In this manner, a variety ofdifferent types of analytes may be detecting using a variety ofdifferent detection system enclosures. Each of the detection systemenclosures may include different sensor arrays mounted within theirchambers. Instead of having to exchange the sensor array for differenttypes of analysis, the entire detection system enclosure may beexchanged. This may prove advantageous, when a variety of detectionschemes are used. For example a first detection system enclosure may beconfigured for white light applications. The first detection systemenclosure may include a white light source, a sensor that includesparticles that produce a visible light response in the presence of ananalyte, and a detector sensitive to white light. A second detectionsystem enclosure may be configured for fluorescent applications,including a fluorescent light source, a sensor array which includesparticles which produce a fluorescent response on the presence of ananalyte, and a fluorescent detector. The second detection systemenclosure may also include other components necessary for producing aproper detection system. For example, the second detection system mayalso include a filter for preventing short wavelength excitation fromproducing “false” signals in the optical detection system duringfluorescence measurements. A user need only select the proper detectionsystem enclosure for the detection of the desired analyte. Since eachdetection system enclosure includes many of the required components, auser does not have to make light source selections, sensor arrayselections or detector arrangement selections to produce a viabledetection system.

In another embodiment, the individual components of the system may beinterchangeable. The system may include coupling members 573 and 575that allow the light source and the detector, respectively, to beremoved from the chamber 550. This may allow a more modular design ofthe system. For example, an analysis may be first performed with a whitelight source to give data corresponding to an absorbance/reflectanceanalysis. After this analysis is performed the light source may bechanged to a ultraviolet light source to allow ultraviolet analysis ofthe particles. Since the particles have already been treated with thefluid, the analysis may be preformed without further treatment of theparticles with a fluid. In this manner a variety of tests may beperformed using a single sensor array.

In one embodiment, the supporting member is made of any material capableof supporting the particles, while allowing the passage of theappropriate wavelength of light. The supporting member may also be madeof a material substantially impervious to the fluid in which the analyteis present. A variety of materials may be used including plastics (e.g.,photoresist materials, acrylic polymers, carbonate polymers, etc.),glass, silicon based materials (e.g., silicon, silicon dioxide, siliconnitride, etc.) and metals. In one embodiment, the supporting memberincludes a plurality of cavities. The cavities are preferably formedsuch that at least one particle is substantially contained within thecavity. Alternatively, a plurality of particles may be contained withina single cavity.

In some embodiments, it will be necessary to pass liquids over thesensor array. The dynamic motion of liquids across the sensor array maylead to displacement of the particles from the cavities. In anotherembodiment, the particles are preferably held within cavities formed ina supporting member by the use of a transmission electron microscope(“TEM”) grid. As depicted in FIG. 19, a cavity 580 is formed in asupporting member 582. After placement of a particle 584 within thecavity, a TEM grid 586 may be placed atop the supporting member 582 andsecured into position. TEM grids and adhesives for securing TEM grids toa support are commercially available from Ted Pella, Inc., Redding,Calif. The TEM grid 586 may be made from a number of materialsincluding, but not limited to, copper, nickel, gold, silver, aluminum,molybdenum, titanium, nylon, beryllium, carbon, and beryllium-copper.The mesh structure of the TEM grid may allow solution access as well asoptical access to the particles that are placed in the cavities. FIG. 20further depicts a top view of a sensor array with a TEM grid 586 formedupon the upper surface of the supporting member 582. The TEM grid 586may be placed on the upper surface of the supporting member, trappingparticles 584 within the cavities 580. As depicted, the openings 588 inthe TEM grid 586 may be sized to hold the particles 584 within thecavities 580, while allowing fluid and optical access to cavities 580.

In another embodiment, a sensor array includes a supporting memberconfigured to support the particles, while allowing the passage of theappropriate wavelength of light to the particle. The supporting member,in one embodiment, includes a plurality of cavities. The cavities may beformed such that at least one particle is substantially contained withinthe cavity. The supporting member may be configured to substantiallyinhibit the displacement of the particles from the cavities during use.The supporting member may also be configured to allow the passage of thefluid through cavities, e.g., the fluid may flow from the top surface ofthe supporting member, past the particle, and out the bottom surface ofthe supporting member. This may increase the contact time between theparticle and the fluid.

FIGS. 21A-G depict a sequence of processing steps for the formation of asilicon based supporting member which includes a removable top cover andbottom cover. The removable top cover may be configured to allow fluidsto pass through the top cover and into the cavity. The removable bottomcover may also be configured to allow the fluid to pass through thebottom cover and out of the cavity. As depicted in FIG. 21A, a series oflayers may be deposited upon both sides of a silicon substrate 610.First removable layers 612 may be deposited upon the silicon substrate.The removable layers 612 may be silicon dioxide, silicon nitride, orphotoresist material. In one embodiment, a layer of silicon dioxide 612is deposited upon both surfaces of the silicon substrate 610. Upon theseremovable layers, covers 614 may be formed. In one embodiment, covers614 are formed from a material that differs from the material used toform the removable layers 612 and which is substantially transparent tothe light source of a detection system. For example, if the removablelayers 612 are formed from silicon dioxide, the cover may be formed fromsilicon nitride. Second removable layers 616 may be formed upon thecovers 614. Second removable layers 616 may be formed from a materialthat differs from the material used to form the covers 614. Secondremovable layers 616 may be formed from a material similar to thematerial used to form the first removable layers 612. In one embodiment,first and second removable layers 612 and 616 are formed from silicondioxide and covers 614 are formed from silicon nitride. The layers arepatterned and etched using standard photolithographic techniques. In oneembodiment, the remaining portions of the layers are substantiallyaligned in the position where the cavities are to be formed in thesilicon substrate 610.

After the layers have been etched, spacer structures may be formed onthe sidewalls of the first removable layers 612, the covers 614, and thesecond removable layers 616, as depicted in FIG. 21B. The spacerstructures may be formed from the same material used to form the secondremovable layers 616. In one embodiment, depositing a spacer layer ofthe appropriate material and subjecting the material to an anisotropicetch may form the spacer structures. An anisotropic etch, such as aplasma etch, employs both physical and chemical removal mechanisms. Ionsare typically bombarded at an angle substantially perpendicular to thesemiconductor substrate upper surface. This causes substantiallyhorizontal surfaces to be removed faster than substantially verticalsurfaces. During this etching procedure the spacer layers are preferablyremoved such that the only regions of the spacer layers that remain maybe those regions near substantially vertical surfaces, e.g., spacerstructures 618.

After formation of the spacer structures 618, cover support structures620, depicted in FIG. 21C, may be formed. The cover support structuresmay be initially formed by depositing a support structure layer upon thesecond removable layer 616 and spacer structures 618. The supportstructure layer is then patterned and etched, using standardphotolithography, to form the support structures 620. In one embodiment,the support structures are formed from a material that differs from theremovable layers material. In one embodiment, the removable layers maybe formed from silicon dioxide while the support structures and coversmay be formed from silicon nitride.

Turning to FIG. 21 D, the second removable layers 616 and an upperportion of the spacer structures 618 are preferably removed using a wetetch process. Removal of the second removable layers leaves the topsurface of the covers 614 exposed. This allows the covers to bepatterned and etched such that openings 622 are formed extending throughthe covers. These openings 622 may be formed in the covers 614 to allowthe passage of fluid through the cover layers. In one embodiment, theopenings 622 are formed to allow fluid to pass through, while inhibitingdisplacement of the particles from the subsequently formed cavities.

After the openings 622 have been formed, the remainder of the firstremovable layers 612 and the remainder of the spacer structures 618 maybe removed using a wet etch. The removal of the removable layers and thespacer structures creates “floating” covers 614, as depicted in FIG.21E. The covers 614 may be held in proximity to the silicon substrate610 by the support structures 620. The covers 614 may now be removed bysliding the covers away from the support structures 620. In this mannerremovable covers 614 may be formed.

After the covers 614 are removed, cavities 640 may be formed in thesilicon substrate 610, as depicted in FIG. 21F. The cavities 640 may beformed by, initially patterning and etching a photoresist material 641to form a masking layer. After the photoresist material 641 ispatterned, the cavities 640 may be etched into the silicon substrate 610using a hydroxide etch, as described previously.

After the cavities 640 are formed, the photoresist material may beremoved and particles 642 may be placed within the cavities, as depictedin FIG. 21G. The particles 642, may be inhibited from being displacedfrom the cavity 640 by placing covers 614 back onto the upper and lowerfaces of the silicon substrate 610.

In another embodiment, a sensor array may be formed using a supportingmember, a removable cover, and a secured bottom layer. FIGS. 22 A-Gdepict a series of processing steps for the formation of a silicon basedsupporting member which includes a removable top cover and a securedbottom layer. The removable top cover is preferably configured to allowfluids to pass through the top cover and into the cavity. As depicted inFIG. 22A, a series of layers may be deposited upon both sides of asilicon substrate 610. A first removable layer 612 may be deposited uponthe upper face 611 of the silicon substrate 610. The removable layer 612may be silicon dioxide, silicon nitride, or photoresist material. In oneembodiment, a layer of silicon dioxide 612 is deposited upon the siliconsubstrate 610. A cover 614 may be formed upon the removable layer 612 ofthe silicon substrate 610. In one embodiment, the cover 614 is formedfrom a material that differs from the material used to form theremovable layer 612 and is substantially transparent to the light sourceof a detection system. For example, if the removable layer 612 is formedfrom silicon dioxide, the cover layer 614 may be formed from siliconnitride. In one embodiment, a bottom layer 615 is formed on the bottomsurface 613 of the silicon substrate 610. In one embodiment, the bottomlayer 615 is formed from a material that is substantially transparent tothe light source of a detection system. A second removable layer 616 maybe formed upon the cover 614. Second removable layer 616 may be formedfrom a material that differs from the material used to form the coverlayer 614. Second removable layer 616 may be formed from a materialsimilar to the material used to form the first removable layer 612. Inone embodiment, first and second removable layers 612 and 616 are formedfrom silicon dioxide and cover 614 is formed from silicon nitride. Thelayers formed on the upper surface 611 of the silicon substrate may bepatterned and etched using standard photolithographic techniques. In oneembodiment, the remaining portions of the layers formed on the uppersurface are substantially aligned in the position where the cavities areto be formed in the silicon substrate 610.

After the layers have been etched, spacer structures may be formed onthe side walls of the first removable layer 612, the cover 614, and thesecond removable layer 616, as depicted in FIG. 22B. The spacerstructures may be formed from the same material used to form the secondremovable layer 616. In one embodiment, the spacer structures may beformed by depositing a spacer layer of the appropriate material andsubjecting the spacer layer to an anisotropic etch. During this etchingprocedure the spacer layer is preferably removed such that the onlyregions of the spacer layer which remain may be those regions nearsubstantially vertical surfaces, e.g., spacer structures 618.

After formation of the spacer structures 618, cover support structures620, depicted in FIG. 22C, may be formed upon the removable layer 616and the spacer structures 618. The cover support structures 620 may beformed by depositing a support structure layer upon the second removablelayer 616 and spacer structures 618. The support structure layer is thenpatterned and etched, using standard photolithography, to form thesupport structures 620. In one embodiment, the support structures areformed from a material that differs from the removable layer materials.In one embodiment, the removable layers may be formed from silicondioxide while the support structures and cover may be formed fromsilicon nitride.

Turning to FIG. 22 D, the second removable layer 616 and an upperportion of the spacer structures 618 may be removed using a wet etchprocess. Removal of the second removable layer leaves the top surface ofthe cover 614 exposed. This allows the cover 614 to be patterned andetched such that openings 622 are formed extending through the cover614. These openings 622 may be formed in the cover 614 to allow thepassage of fluid through the cover. In one embodiment, the openings 622are formed to allow fluid to pass through, while inhibiting displacementof the particle from a cavity. The bottom layer 615 may also besimilarly patterned and etched such that openings 623 may be formedextending thorough the bottom layer 615.

After the openings 622 and 623 are formed, the first removable layer 612and the remainder of the spacer structures 618 may be removed using awet etch. The removal of the removable layers and the spacer structurescreates a “floating” cover 614, as depicted in FIG. 22E. The cover 614may be held in proximity to the silicon substrate 610 by the supportstructures 620. The cover 614 may now be removed by sliding the cover614 away from the support structures 620. In this manner a removablecover 614 may be formed.

After the cover 614 is removed, cavities 640 may be formed in thesilicon substrate 610, as depicted in FIG. 22F. The cavities 640 may beformed by, initially patterning and etching a photoresist material 641to form a masking layer. After the photoresist material 614 ispatterned, the cavities 640 may be etched into the silicon substrate 610using a hydroxide etch, as described previously.

After the cavities 640 are formed, the photoresist material may beremoved and particles 642 may be placed within the cavities, as depictedin FIG. 22G. The particles 642, may be inhibited from being displacedfrom the cavity 640 by placing cover 614 back onto the upper face 611 ofthe silicon substrate 610. The bottom layer 615 may also aid ininhibiting the particle 642 from being displaced from the cavity 640.Openings 622 in cover 614 and openings 623 in bottom layer 615 may allowfluid to pass through the cavity during use.

In another embodiment, a sensor array may be formed using a supportingmember and a removable cover. FIGS. 23A-G depict a series of processingsteps for the formation of a silicon based supporting member whichincludes a removable cover. The removable cover is preferably configuredto allow fluids to pass through the cover and into the cavity. Asdepicted in FIG. 23A, a series of layers may be deposited upon the uppersurface 611 of a silicon substrate 610. A first removable layer 612 maybe deposited upon the upper face 611 of the silicon substrate 610. Theremovable layer 612 may be silicon dioxide, silicon nitride, orphotoresist material. In one embodiment, a layer of silicon dioxide 612is deposited upon the silicon substrate 610. A cover 614 may be formedupon the removable layer 612. In one embodiment, the cover is formedfrom a material which differs from the material used to form theremovable layer 612 and which is substantially transparent to the lightsource of a detection system. For example, if the removable layer 612 isformed from silicon dioxide, the cover 614 may be formed from siliconnitride. A second removable layer 616 may be formed upon the cover 614.Second removable layer 616 may be formed from a material that differsfrom the material used to form the cover 614. Second removable layer 616may be formed from a material similar to the material used to form thefirst removable layer 612. In one embodiment, first and second removablelayers 612 and 616 are formed from silicon dioxide and cover 614 isformed from silicon nitride. The layers formed on the upper surface 611of the silicon substrate may be patterned and etched using standardphotolithographic techniques. In one embodiment, the remaining portionsof the layers formed on the upper surface are substantially aligned inthe position where the cavities are to be formed in the siliconsubstrate 610.

After the layers have been etched, spacer structures 618 may be formedon the side walls of the first removable layer 612, the cover layer 614,and the second removable layer 616, as depicted in FIG. 23B. The spacerstructures 618 may be formed from the same material used to form thesecond removable layer 616. In one embodiment, the spacers may be formedby depositing a spacer layer of the appropriate material upon the secondremovable layer and subjecting the material to an anisotropic etch.During this etching procedure the spacer layer is preferably removedsuch that the only regions of the spacer layer which remain may be thoseregions near substantially vertical surfaces, e.g., spacer structures618.

After formation of the spacer structures 618, cover support structures620, depicted in FIG. 23C, may be formed upon the removable layer 616and the spacer structures 618. The cover support structure may be formedby initially depositing a support structure layer upon the secondremovable layer 616 and spacer structures 618. The support structurelayer is then patterned and etched, using standard photolithography, toform the support structures 620. In one embodiment, the supportstructures 620 are formed from a material that differs from theremovable layer materials. In one embodiment, the removable layers maybe formed from silicon dioxide while the support structure and coverlayer may be formed from silicon nitride.

Turning to FIG. 23D, the second removable layer 616 and an upper portionof the spacer structures 618 may be removed using a wet etch process.Removal of the second removable layer leaves the top surface of thecover 614 exposed. This allows the cover 614 to be patterned and etchedsuch that openings 622 are formed extending through the cover 614. Theseopenings 622 may be formed in the cover 614 to allow the passage offluid through the cover 614.

After the openings 622 are formed, the remainder of the first removablelayer 612 and the remainder of the spacer structures 618 may be removedusing a wet etch. The removal of the removable layers and the spacerstructures creates a “floating” cover 614, as depicted in FIG. 23E. Thecover 614 is preferably held in proximity to the silicon substrate 610by the support structures 620. The cover 614 may now be removed bysliding the cover 614 away from the support structures 620. In thismanner a removable cover 614 may be formed.

After the cover 614 is removed, cavities 640 may be formed in thesilicon substrate 610, as depicted in FIG. 23F. The cavities 640 may beformed by initially depositing and patterning a photoresist material 641upon the silicon support 610. After the photoresist material 614 ispatterned, the cavities 640 may be etched into the silicon substrate 610using a hydroxide etch, as described previously. The etching of thecavities may be accomplished such that a bottom width of the cavity 643is less than a width of a particle 642. In one embodiment, the width ofthe bottom of the cavity may be controlled by varying the etch time.Typically, longer etching times result in a larger opening at the bottomof the cavity. By forming a cavity in this manner, a particle placed inthe cavity may be too large to pass through the bottom of the cavity.Thus, a supporting member that does not include a bottom layer may beformed. An advantage of this process is that the processing steps may bereduced making production simpler.

After the cavities 640 are formed, the photoresist material may beremoved and particles 642 may be placed within the cavities, as depictedin FIG. 23G. The particles 642, may be inhibited from being displacedfrom the cavity 640 by placing cover 614 back onto the upper face 611 ofthe silicon substrate 610. The narrow bottom portion of the cavity mayalso aid in inhibiting the particle 642 from being displaced from thecavity 640.

FIGS. 24A-d depict a sequence of processing steps for the formation of asilicon based supporting member which includes a top partial cover and abottom partial cover. The top partial cover and bottom partial coversare, in one embodiment, configured to allow fluids to pass into thecavity and out through the bottom of the cavity. As depicted in FIG.24A, a bottom layer 712 may be deposited onto the bottom surface of asilicon substrate 710. The bottom layer 712 may be silicon dioxide,silicon nitride, or photoresist material. In one embodiment, a layer ofsilicon nitride 712 is deposited upon the silicon substrate 710. In oneembodiment, openings 714 are formed through the bottom layer as depictedin FIG. 24A. Openings 714, in one embodiment, are substantially alignedwith the position of the cavities to be subsequently formed. Theopenings 714 may have a width that is substantially less than a width ofa particle. Thus a particle will be inhibited from passing through theopenings 714.

Cavities 716 may be formed in the silicon substrate 710, as depicted inFIG. 24B. The cavities 716 may be formed by initially depositing andpatterning a photoresist layer upon the silicon substrate 710. After thephotoresist material is patterned, cavities 716 may be etched into thesilicon substrate 710 using a number of etching techniques, includingwet and plasma etches.

The width of the cavities 716 is preferably greater than the width of aparticle, thus allowing a particle to be placed within each of thecavities. The cavities 716, in one embodiment, are preferably formedsuch that the cavities are substantially aligned over the openings 714formed in the bottom layer.

After the cavities have been formed, particles 718 may be inserted intothe cavities 716, as depicted in FIG. 24C. The etched bottom layer 712may serve as a support for the particles 718. Thus the particles 718 maybe inhibited from being displaced from the cavities by the bottom layer712. The openings 714 in the bottom layer 712 may allow fluid to passthrough the bottom layer during use.

After the particles are placed in the cavities, a top layer 720 may beplaced upon the upper surface 717 of the silicon substrate. In oneembodiment, the top layer 720 is formed from a material is substantiallytransparent to the light source of a detection system. The top layer maybe formed from silicon nitride, silicon dioxide or photoresist material.In one embodiment, a sheet of photoresist material is used. After thetop layer 620 is formed, openings 719 may be formed in the top layer toallow the passage of the fluid into the cavities. If the top layer 720is composed of photoresist material, after depositing the photoresistmaterial across the upper surface of the silicon substrate, the openingsmay be initially formed by exposing the photoresist material to theappropriate wavelength and pattern of light. If the top layer is composeof silicon dioxide or silicon nitride the top layer 720 may be developedby forming a photoresist layer upon the top layer, developing thephotoresist, and using the photoresist to etch the underlying top layer.

Similar sensor arrays may be produced using materials other than siliconfor the supporting member. For example, as depicted in FIG. 25 A-D, thesupporting member may be composed of photoresist material. In oneembodiment, sheets of photoresist film may be used to form thesupporting member. Photoresist film sheets are commercially availablefrom E. I. du Pont de Nemours and Company, Wilmington, Del. under thecommercial name RISTON. The sheets come in a variety of sizes, the mostcommon having a thickness ranging from about 1 mil. (25 μm) to about 2mil. (50 μm).

In an embodiment, a first photoresist layer 722 is developed and etchedsuch that openings 724 are formed. The openings may be formed proximatethe location of the subsequently formed cavities. Preferably, theopenings have a width that is substantially smaller than a width of theparticle. The openings may inhibit displacement of the particle from acavity. After the first photoresist layer 720 is patterned and etched, amain layer 726 is formed upon the bottom layer. The main layer 720 ispreferably formed from a photoresist film that has a thicknesssubstantially greater than a typical width of a particle. Thus, if theparticles have a width of about 30 μm, a main layer may be composed of a50 μm photoresist material. Alternatively, the photoresist layer may becomposed of a multitude of photoresist layers placed upon each otheruntil the desired thickness is achieved, as will be depicted in laterembodiments.

The main photoresist layer may be patterned and etched to form thecavities 728, as depicted in FIG. 25B. The cavities, in one embodiment,are substantially aligned above the previously formed openings 724.Cavities 728, in one embodiment, have a width which is greater than awidth of a particle.

For many types of analysis, the photoresist material is substantiallytransparent to the light source used. Thus, as opposed to a siliconsupporting member, the photoresist material used for the main supportinglayer may be substantially transparent to the light used by the lightsource. In some circumstances, the transparent nature of the supportingmember may allow light from the cavity to migrate, through thesupporting member, into a second cavity. This leakage of light from onecavity to the next may lead to detection problems. For example, if afirst particle in a first cavity produces a fluorescent signal inresponse to an analyte, this signal may be transmitted through thesupporting member and detected in a proximate cavity. This may lead toinaccurate readings for the proximately spaced cavities, especially if aparticularly strong signal is produced by the interaction of theparticle with an analyte.

To reduce the occurrence of this “cross-talk”, a substantiallyreflective layer 730 may be formed along the inner surface of thecavity. In one embodiment, the reflective layer 730 is composed of ametal layer which is formed on the upper surface of the main layer andthe inner surface of the cavity. The metal layer may be deposited usingchemical vapor deposition or other known techniques for depositing thinmetal layers. The presence of a reflective layer may inhibit“cross-talk” between the cavities.

After the cavities 728 have been formed, particles 718 may be insertedinto the cavities 728, as depicted in FIG. 25C. The first photoresistlayer 722 may serve as a support for the particles 718. The particlesmay be inhibited from being displaced from the cavities by the firstphotoresist layer 722. The openings 724 in the first photoresist layer722 may allow fluid to pass through the bottom layer during use.

After the particles 728 are placed in the cavities 728, a topphotoresist layer 732 may be placed upon the upper surface of thesilicon substrate. After the cover layer is formed, openings 734 may beformed in the cover layer to allow the passage of the fluid into thecavities.

In another embodiment, the supporting member may be formed from aplastic substrate, as depicted in FIG. 26A-D. In one embodiment, theplastic substrate is composed of a material which is substantiallyresistant to the fluid which includes the analyte. Examples of plasticmaterials which may be used to form the plastic substrate include, butare not limited to, acrylic resins, polycarbonates, polyester resins,polyethylenes, polyimides, polyvinyl polymers (e.g., polyvinyl chloride,polyvinyl acetate, polyvinyl dichloride, polyvinyl fluoride, etc.),polystyrenes, polypropylenes, polytetrafluoroethylenes, andpolyurethanes. The plastic substrate may be substantially transparent orsubstantially opaque to the light produced by the light source. Afterobtaining a suitable plastic material 740, a series of cavities 742 maybe formed in the plastic material. The cavities 740 may be formed bydrilling (either mechanically or with a laser), transfer molding (e.g.,forming the cavities when the plastic material is formed usingappropriately shaped molds), or using a punching apparatus to punchcavities into the plastic material. In one embodiment, the cavities 740are formed such that a lower portion 743 of the cavities issubstantially narrower than an upper portion 744 of the cavities. Thelower portion 743 of the cavities may have a width substantially lessthan a width of a particle. The lower portion 743 of the cavities 740may inhibit the displacement of a particle from the cavity 740. Whiledepicted as rectangular, with a narrower rectangular opening at thebottom, it should be understood that the cavity may be formed in anumber of shapes including but not limited to pyramidal, triangular,trapezoidal, and oval shapes. An example of a pyramidal cavity which istapered such that the particle is inhibited from being displaced fromthe cavity is depicted in FIG. 25D.

After the cavities 742 are formed, particles 718 may be inserted intothe cavities 742, as depicted in FIG. 26B. The lower portion 743 of thecavities may serve as a support for the particles 718. The particles 718may be inhibited from being displaced from the cavities 742 by the lowerportion 743 of the cavity. After the particles are placed in thecavities 740, a cover 744 may be placed upon the upper surface 745 ofthe plastic substrate 740, as depicted in FIG. 26C.

In one embodiment, the cover is formed from a film of photoresistmaterial. After the cover 744 is placed on the plastic substrate 740,openings 739 may be formed in the cover layer to allow the passage ofthe fluid into the cavities. In some circumstances a substantiallytransparent plastic material may be used. As described above, the use ofa transparent supporting member may lead to “cross-talk” between thecavities. To reduce the occurrence of this “cross-talk”, a substantiallyreflective layer 748 may be formed on the inner surface 746 of thecavity, as depicted in FIG. 26E. In one embodiment, the reflective layer748 is composed of a metal layer which is formed on the inner surface ofthe cavities 742. The metal layer may be deposited using chemical vapordeposition or other techniques for depositing thin metal layers. Thepresence of a reflective layer may inhibit cross-talk between thecavities.

In another embodiment, a silicon based supporting member for a sensingparticle may be formed without a bottom layer. In this embodiment, thecavity may be tapered to inhibit the passage of the particle from thecavity, through the bottom of the supporting member. FIG. 27A-D, depictsthe formation of a supporting member from a silicon substrate. In thisembodiment, a photoresist layer 750 is formed upon an upper surface of asilicon substrate 752, as depicted in FIG. 27A. The photoresist layer750 may be patterned and developed such that the regions of the siliconsubstrate in which the cavities will be formed are exposed.

Cavities 754 may now be formed, as depicted in FIG. 27B, by subjectingthe silicon substrate to an anisotropic etch. In one embodiment, apotassium hydroxide etch is used to produced tapered cavities. Theetching may be controlled such that the width of the bottom of thecavities 750 is less than a width of the particle. After the cavitieshave been etched, a particle 756 may be inserted into the cavities 754as depicted in FIG. 27C. The particle 756 may be inhibited from passingout of the cavities 754 by the narrower bottom portion of the cavities.After the particle is positioned within the cavities 754, a cover 758may be formed upon the silicon substrate 752, as depicted in FIG. 27D.The cover may be formed of any material substantially transparent to thelight produced by the light source used for analysis. Openings 759 maybe formed in the cover 758 to allow the fluid to pass into the cavityfrom the top face of the supporting member 752. The openings 759 in thecover and the opening at the bottom of the cavities 754 together mayallow fluid to pass through the cavity during use.

In another embodiment, a supporting member for a sensing particle may beformed from a plurality of layers of a photoresist material. In thisembodiment, the cavity may be tapered to inhibit the passage of theparticle from the cavity, through the bottom of the supporting member.FIGS. 28A-E depict the formation of a supporting member from a pluralityof photoresist layers. In an embodiment, a first photoresist layer 760is developed and etched to form a series of openings 762 which arepositioned at the bottom of subsequently formed cavities, as depicted inFIG. 28A. As depicted in FIG. 28B, a second layer of photoresistmaterial 764 may be formed upon the first photoresist layer 760. Thesecond photoresist layer may be developed and etched to form openingssubstantially aligned with the openings of the first photoresist layer760. The openings formed in the second photoresist layer 764, in oneembodiment, are substantially larger than the layers formed in the firstphotoresist layer 760. In this manner, a tapered cavity may be formedwhile using multiple photoresist layers.

As depicted in FIG. 28C, additional layers of photoresist material 766and 768 may be formed upon the second photoresist layer 764. Theopenings of the additional photoresist layers 766 and 768 may beprogressively larger as each layer is added to the stack. In thismanner, a tapered cavity may be formed. Additional layers of photoresistmaterial may be added until the desired thickness of the supportingmember is obtained. The thickness of the supporting member, in oneembodiment, is greater than a width of a particle. For example, if alayer of photoresist material has a thickness of about 25 μm and aparticle has a width of about 100 μm, a supporting member may be formedfrom four or more layers of photoresist material. While depicted aspyramidal, the cavity may be formed in a number of different shapes,including but not limited to, rectangular, circular, oval, triangular,and trapezoidal. Any of these shapes may be obtained by appropriatepatterning and etching of the photoresist layers as they are formed.

In some instances, the photoresist material may be substantiallytransparent to the light produced by the light source. As describedabove, the use of a transparent supporting member may lead to“cross-talk” between the cavities. To reduce the occurrence of this“cross-talk”, a substantially reflective layer 770 may be formed alongthe inner surface of the cavities 762, as depicted in FIG. 28D. In oneembodiment, the reflective layer is composed of a metal layer which isformed on the inner surface of the cavities 762. The metal layer may bedeposited using chemical vapor deposition or other techniques fordepositing thin metal layers. The presence of a reflective layer mayinhibit “cross-talk” between the cavities.

After the cavities 762 are formed, particles 772 may be inserted intothe cavities 762, as depicted in FIG. 28D. The narrow portions of thecavities 762 may serve as a support for the particles 772. The particles772 may be inhibited from being displaced from the cavities 762 by thelower portion of the cavities. After the particles 772 are placed in thecavities 762, a cover 774 may be placed upon the upper surface of thetop layer 776 of the supporting member, as depicted in FIG. 28E. In oneembodiment, the cover 774 is also formed from a film of photoresistmaterial. After the cover layer is formed, openings 778 may be formed inthe cover 774 to allow the passage of the fluid into the cavities.

In another embodiment, a supporting member for a sensing particle may beformed from photoresist material which includes a particle supportlayer. FIGS. 29A-E depict the formation of a supporting member from aseries of photoresist layers. In an embodiment, a first photoresistlayer 780 is developed and etched to form a series of openings 782 whichmay become part of subsequently formed cavities. In another embodiment,a cavity having the appropriate depth may be formed by forming multiplelayers of a photoresist material, as described previously. As depictedin FIG. 29B, a second photoresist layer 784 may be formed upon the firstphotoresist layer 780. The second photoresist layer 784 may be patternedto form openings substantially aligned with the openings of the firstphotoresist layer 782. The openings formed in the second photoresistlayer 784 may be substantially equal in size to the previously formedopenings. Alternatively, the openings may be variable in size to formdifferent shaped cavities.

For reasons described above, a substantially reflective layer 786 may beformed along the inner surface of the cavities 782 and the upper surfaceof the second photoresist layer 784, as depicted in FIG. 29C. In oneembodiment, the reflective layer is composed of a metal layer. The metallayer may be deposited using chemical vapor deposition or othertechniques for depositing thin metal layers. The presence of areflective layer may inhibit “cross-talk” between the cavities.

After the metal layer is deposited, a particle support layer 788 may beformed on the bottom surface of the first photoresist layer 780, asdepicted in FIG. 29D. The particle support layer 788 may be formed fromphotoresist material, silicon dioxide, silicon nitride, glass or asubstantially transparent plastic material. The particle support layer788 may serve as a support for the particles placed in the cavities 782.The particle support layer, in one embodiment, is formed from a materialthat is substantially transparent to the light produced by the lightsource.

After the particle supporting layer 788 is formed, particles 785 may beinserted into the cavities 782, as depicted in FIG. 29E. The particlesupport layer 788 may serve as a support for the particles. Thus theparticles 785 may be inhibited from being displaced from the cavities bythe particle support layer 788. After the particles 785 are placed inthe cavities 782, a cover 787 may be placed upon the upper surface ofthe second photoresist layer 784, as depicted in FIG. 29E. In oneembodiment, the cover is also formed from a film of photoresistmaterial. After the cover is formed, openings 789 may be formed in thecover 787 to allow the passage of the fluid into the cavities. In thisembodiment, the fluid is inhibited from flowing through the supportingmember. Instead, the fluid may flow into and out of the cavities via theopenings 789 formed in the cover 787.

A similar supporting member may be formed from a plastic material, asdepicted in FIGS. 30A-D. The plastic material may be substantiallyresistant to the fluid which includes the analyte. The plastic materialmay be substantially transparent or substantially opaque to the lightproduced by the light source. After obtaining a suitable plasticsubstrate 790, a series of cavities 792 may be formed in the plasticsubstrate 790. The cavities may be formed by drilling (eithermechanically or with a laser), transfer molding (e.g., forming thecavities when the plastic substrate is formed using appropriately shapedmolds), or using a punching machine to form the cavities. In oneembodiment, the cavities extend through a portion of the plasticsubstrate, terminating proximate the bottom of the plastic substrate,without passing through the plastic substrate. After the cavities 792are formed, particles 795 may be inserted into the cavities 792, asdepicted in FIG. 30B. The bottom of the cavity may serve as a supportfor the particles 795. After the particles are placed in the cavities, acover 794 may be placed upon the upper surface of the plastic substrate790, as depicted in FIG. 30C. In one embodiment, the cover may be formedfrom a film of photoresist material. After the cover 794 is formed,openings 796 may be formed in the cover to allow the passage of thefluid into the cavities. While depicted as rectangular, is should beunderstood that the cavities may be formed in a variety of differentshapes, including triangular, pyramidal, pentagonal, polygonal, oval, orcircular. It should also be understood that cavities having a variety ofdifferent shapes may be formed into the same plastic substrate, asdepicted in FIG. 30D.

In one embodiment, a series of channels may be formed in the supportingmember interconnecting some of the cavities, as depicted in FIG. 3.Pumps and valves may also be incorporated into the supporting member toaid passage of the fluid through the cavities. A schematic figure of adiaphragm pump 800 is depicted in FIG. 31. Diaphragm pumps, in general,include a cavity 810, a flexible diaphragm 812, an inlet valve 814, andan outlet valve 816. The flexible diaphragm 812, during use, isdeflected as shown by arrows 818 to create a pumping force. As thediaphragm is deflected toward the cavity 810 it may cause the inletvalve 814 to close, the outlet valve 816 to open and any liquid which isin the cavity 810 will be forced toward the outlet 816. As the diaphragmmoves away from the cavity 810, the outlet valve 816 may be pulled to aclosed position, and the inlet valve 814 may be opened, allowingadditional fluid to enter the cavity 810. In this manner a pump may beused to pump fluid through the cavities. It should be understood thatthe pump depicted in FIG. 31 is a generalized version of a diaphragmbased pump. Actual diaphragm pumps may have different shapes or may haveinlet and outlet valves which are separate from the pumping device.

In one embodiment, the diaphragm 810 may be made from a piezoelectricmaterial. This material will contract or expand when an appropriatevoltage is applied to the diaphragm. Pumps using a piezoelectricdiaphragms are described in U.S. Pat. Nos. 4,344,743, 4,938,742,5,611,676, 5,705,018, and 5,759,015, all of which are incorporatedherein by reference. In other embodiments, the diaphragm may beactivated using a pneumatic system. In these systems, an air system maybe coupled to the diaphragm such that changes in air density about thediaphragm, induced by the pneumatic system, may cause the diaphragm tomove toward and away from the cavity. A pneumatically controlled pump isdescribed in U.S. Pat. No. 5,499,909 which is incorporated herein byreference. The diaphragm may also be controlled using a heat activatedmaterial. The diaphragm may be formed from a temperature sensitivematerial. In one embodiment, the diaphragm may be formed from a materialwhich is configured to expand and contract in response to temperaturechanges. A pump system which relies on temperature activated diaphragmis described in U.S. Pat. No. 5,288,214 which is incorporated herein byreference.

In another embodiment, an electrode pump system may be used. FIG. 32depicts a typical electrode based system. A series of electrodes 820 maybe arranged along a channel 822 which may lead to a cavity 824 whichincludes a particle 826. By varying the voltage in the electrodes 820 acurrent flow may be induced in the fluid within the channel 822.Examples of electrode based systems include, but are not limited to,electroosmosis systems, electrohydrodynamic systems, and combinations ofelectroosmosis and electrohydrodynamic systems.

Electrohydrodynamic pumping of fluids is known and may be applied tosmall capillary channels. In an electrohydrodynamic system electrodesare typically placed in contact with the fluid when a voltage isapplied. The applied voltage may cause a transfer in charge either bytransfer or removal of an electron to or from the fluid. This electrontransfer typically induces liquid flow in the direction from thecharging electrode to the oppositely charged electrode.Electrohydrodynamic pumps may be used for pumping fluids such as organicsolvents.

Electroosmosis, is a process which involves applying a voltage to afluid in a small space, such as a capillary channel, to cause the fluidto flow. The surfaces of many solids, including quartz, glass and thelike, become variously charged, negatively or positively, in thepresence of ionic materials, such as for example salts, acids or bases.The charged surfaces will attract oppositely charged (positive ornegative) counterions in aqueous solutions. The application of a voltageto such a solution results in a migration of the counterions to theoppositely charged electrode, and moves the bulk of the fluid as well.The volume flow rate is proportional to the current, and the volume flowgenerated in the fluid is also proportional to the applied voltage. Anelectroosmosis pump system is described in U.S. Pat. No. 4,908,112 whichis incorporated herein by reference.

In another embodiment, a combination of electroosmosis pumps andelectrohydrodynamic pumps may be used. Wire electrodes may be insertedinto the walls of a channel at preselected intervals to form alternatingelectroosmosis and electrohydrodynamic devices. Because electroosmosisand electrohydrodynamic pumps are both present, a plurality of differentsolutions, both polar and non-polar, may be pump along a single channel.Alternatively, a plurality of different solutions may be passed along aplurality of different channels connected to a cavity. A system whichincludes a combination of electroosmosis pumps and electrohydrodynamicpumps is described in U.S. Pat. No. 5,632,876 which is incorporatedherein by reference.

In an embodiment, a pump may be incorporated into a sensor array system,as depicted in FIG. 32. A sensor array 830 includes at least one cavity832 in which a particle 834 may be placed. The cavity 832 may beconfigured to allow fluid to pass through the cavity during use. A pump836 may be incorporated onto a portion of the supporting member 838. Achannel 831 may be formed in the supporting member 838 coupling the pump836 to the cavity 832. The channel 831 may be configured to allow thefluid to pass from the pump 836 to the cavity 832. The pump 836 may bepositioned away from the cavity 832 to allow light to be directedthrough the cavity during use. The supporting member 838 and the pump836 may be formed from a silicon substrate, a plastic material, orphotoresist material. The pump 836 may be configured to pump fluid tothe cavity via the channel, as depicted by the arrows in FIG. 32. Whenthe fluid reaches the cavity 832, the fluid may flow past the particle834 and out through the bottom of the cavity. An advantage of usingpumps is that better flow through the channels may be achieved.Typically, the channels and cavities may have a small volume. The smallvolume of the cavity and channel tends to inhibit flow of the fluidthrough the cavity. By incorporating a pump, the flow of fluid to thecavity and through the cavity may be increased, allowing more rapidtesting of the fluid sample. While a diaphragm based pump system isdepicted in FIG. 33, it should be understood that electrode basedpumping systems may also be incorporated into the sensor array toproduce fluid flows.

In another embodiment, a pump may be coupled to a supporting member foranalyzing analytes in a fluid stream, as depicted in FIG. 34. A channel842 may couple a pump 846 to multiple cavities 844 formed in asupporting member 840. The cavities 842 may include sensing particles848. The pump may be configured to create a flow of the fluid throughthe channel 842 to the cavities 848. In one embodiment, the cavities mayinhibit the flow of the fluid through the cavities 844. The fluid mayflow into the cavities 844 and past the particle 848 to create a flow offluid through the sensor array system. In this manner a single pump maybe used to pass the fluid to multiple cavities. While a diaphragm pumpsystem is depicted in FIG. 33, it should be understood that electrodepumping systems may also be incorporated into the supporting member tocreate similar fluid flows.

In another embodiment, multiple pumps may be coupled to a supportingmember of a sensor array system. In one embodiment, the pumps may becoupled in series with each other to pump fluid to each of the cavities.As depicted in FIG. 35, a first pump 852 and a second pump 854 may becoupled to a supporting member 850. The first pump 852 may be coupled toa first cavity 856. The first pump may be configured to transfer fluidto the first cavity 856 during use. The cavity 856 may be configured toallow the fluid to pass through the cavity to a first cavity outletchannel 858. A second pump 854 may also be coupled to the supportingmember 850. The second pump 854 may be coupled to a second cavity 860and the first cavity outlet channel 858. The second pump 854 may beconfigured to transfer fluid from the first cavity outlet channel 858 tothe second cavity 860. The pumps may be synchronized such that a steadyflow of fluid through the cavities is obtained. Additional pumps may becoupled to the second cavity outlet channel 862 such that the fluid maybe pumped to additional cavities. In one embodiment, each of thecavities in the supporting member is coupled to a pump configured topump the fluid stream to the cavity.

In another embodiment, multiple electrode based pumps may beincorporated herein into the sensor array system. The pumps may beformed along the channels which couple the cavities. As depicted in FIG.36, a plurality of cavities 870 may be formed in a supporting member 872of a sensor array. Channels 874 may also be formed in the supportingmember 872 interconnecting the cavities 870 with each other. An inletchannel 876 and an outlet channel 877, which allow the fluid to passinto and out of the sensor array, respectively, may also be formed. Aseries of electrodes 878 may be positioned over the channels 874, 876,and 877. The electrodes may be used to form an electroosmosis pumpingsystem or an electrohydrodynamic pumping system. The electrodes may becoupled to a controller 880 which may apply the appropriate voltage tothe appropriate electrodes to produce a flow of the fluid through thechannels. The pumps may be synchronized such that a steady flow of fluidthrough the cavities is obtained. The electrodes may be positionedbetween the cavities such that the electrodes do not significantlyinterfere with the application of light to the cavities.

In some instances it may be necessary to add a reagent to a particlebefore, during or after an analysis process. Reagents may includereceptor molecules or indicator molecules. Typically, such reagents maybe added by passing a fluid stream which includes the reagent over thesensor array. In an embodiment, the reagent may be incorporated hereininto the sensor array system which includes two particles. In thisembodiment, a sensor array system 900 may include two particles 910 and920 for each sensing position of the sensor array, as depicted in FIG.37. The first particle 910 may be positioned in a first cavity 912. Thesecond particle 920 may be positioned in a second cavity 922. In oneembodiment, the second cavity is coupled to the first cavity via achannel 930. The second particle includes a reagent which is at leastpartially removable from the second particle 920. The reagent may alsobe configured to modify the first particle 910, when the reagent iscontacted with the first particle, such that the first particle willproduce a signal when the first particle interacts with an analyteduring use. The reagent may be added to the first cavity before, duringor after a fluid analysis. The reagent is preferably coupled to thesecond particle 920. The a portion of the reagent coupled to the secondparticle may be decoupled from the particle by passing a decouplingsolution past the second particle. The decoupling solution may include adecoupling agent which will cause at least a portion of the reagent tobe at released by the particle. A reservoir 940 may be formed on thesensor array to hold the decoupling solution.

A first pump 950 and a second pump 960 may also be coupled to thesupporting member 915. The first pump 950 may be configured to pumpfluid from a fluid inlet 952 to the first cavity 912 via channel 930.The fluid inlet 952 is the location where the fluid, which includes theanalyte, is introduced into the sensor array system. A second pump 950may be coupled to the reservoir 940 and the second cavity 922. Thesecond pump 960 may be used to transfer the decoupling solution from thereservoir to the second cavity 922. The decoupling solution may passthrough the second cavity 922 and into first cavity 912. Thus, as thereagent is removed the second particle it may be transferred to thefirst cavity 912, where the reagent may interact with the first particle910. The reservoir may be refilled by removing the reservoir outlet 942,and adding additional fluid to the reservoir 940. While diaphragm basedpump systems are depicted in FIG. 37, it should be understood thatelectrode based pumping systems may also be incorporated herein into thesensor array to produce fluid flows.

The use of such a system is described by way of example. In someinstances it may be desirable to add a reagent to the first particleprior to passing the fluid which includes the analyte to the firstparticle. The reagent may be coupled to the second particle and placedin the sensor array prior to use, typically during construction of thearray. A decoupling solution may be added to the reservoir before use. Acontroller 970 may also be coupled to the system to allow automaticoperation of the pumps. The controller 970 may be configured to initiatethe analysis sequence by activating the second pump 960, causing thedecoupling solution to flow from the reservoir 940 to the second cavity922. As the fluid passes through the second cavity 922, the decouplingsolution may cause at least some of the reagent molecules to be releasedfrom the second particle 920. The decoupling solution may be passed outof the second cavity 922 and into the first cavity 912. As the solutionpasses through the first cavity, some of the reagent molecules may becaptured by the first particle 910. After a sufficient number ofmolecules have been captured by the first particle 910, flow of fluidthorough the second cavity 922 may be stopped. During thisinitialization of the system, the flow of fluid through the first pumpmay be inhibited.

After the system is initialized, the second pump may be stopped and thefluid may be introduced to the first cavity. The first pump may be usedto transfer the fluid to the first cavity. The second pump may remainoff, thus inhibiting flow of fluid from the reservoir to the firstcavity. It should be understood that the reagent solution may be addedto the first cavity while the fluid is added to the first cavity. Inthis embodiment, both the first and second pumps may be operatedsubstantially simultaneously.

Alternatively, the reagent may be added after an analysis. In someinstances, a particle may interact with an analyte such that a change inthe receptors attached to the first particle occurs. This change maynot, however produce a detectable signal. The reagent attached to thesecond bead may be used to produce a detectable signal when it interactswith the first particle, if a specific analyte is present. In thisembodiment, the fluid is introduced into the cavity first. After theanalyte has been given time to react with the particle, the reagent maybe added to the first cavity. The interaction of the reagent with theparticle may produce a detectable signal. For example, an indicatorreagent may react with a particle which has been exposed to an analyteto produce a color change on the particle. Particle which have not beenexposed to the analyte may remain unchanged or show a different colorchange.

As shown in FIG. 1, a system for detecting analytes in a fluid mayinclude a light source 110, a sensor array 120 and a detector 130. Thesensor array 120 is preferably formed of a supporting member which isconfigured to hold a variety of particles 124 in an ordered array. Ahigh sensitivity CCD array may be used to measure changes in opticalcharacteristics which occur upon binding of the biological/chemicalagents. Data acquisition and handling is preferably performed withexisting CCD technology. As described above, calorimetric analysis maybe performed using a white light source and a color CCD detector.However, color CCD detectors are typically more expensive than grayscale CCD detectors.

In one embodiment, a gray scale CCD detector may be used to detectcolorimetric changes. In one embodiment, a gray scale detector may bedisposed below a sensor array to measure the intensity of light beingtransmitted through the sensor array. A series of lights (e.g., lightemitting diodes) may be arranged above the sensor array. In oneembodiment, groups of three LED lights may be arranged above each of thecavities of the array. Each of these groups of LED lights may include ared, blue and a green light. Each of the lights may be operatedindividually such that one of the lights may be on while the other twolights are off. In order to provide color information while using a grayscale detector, each of the lights is sequentially turned on and thegray scale detector is used to measure the intensity of the lightpassing through the sensor array. After information from each of thelights is collected, the information may be processed to derive theabsorption changes of the particle.

In one embodiment, the data collected by the gray scale detector may berecorded using 8 bits of data. Thus, the data will appear as a valuebetween 0 and 255. The color of each chemical sensitive element may berepresented as a red, blue and green value. For example, a blankparticle (i.e., a particle which does not include a receptor) willtypically appear white. When each of the LED lights (red, blue andgreen) are operated the CCD detector will record a value correspondingto the amount of light transmitted through the cavity. The intensity ofthe light may be compared to a blank particle, to determine theabsorbance of a particle with respect to the LED light which is used.Thus, the red, green and blue components may be recorded individuallywithout the use of a color CCD detector. In one embodiment, it is foundthat a blank particle exhibits an absorbance of about 253 whenilluminated with a red LED, a value of about 250 when illuminated by agreen LED, and a value of about 222 when illuminated with a blue LED.This signifies that a blank particle does not significantly absorb red,green or blue light. When a particle with a receptor is scanned, theparticle may exhibit a color change, due to absorbance by the receptor.For example, it was found that when a particle which includes a5-carboxyfluorescein receptor is subjected to white light, the particleshows a strong absorbance of blue light. When a red LED is used toilluminate the particle, the gray scale CCD detector may detect a valueof about 254. When the green LED is used, the gray scale detector maydetect a value of about 218. When a blue LED light is used, a gray scaledetector may detect a value of about 57. The decrease in transmittanceof blue light is believed to be due to the absorbance of blue light bythe 5-carboxyfluorescein. In this manner the color changes of a particlemay be quantitatively characterized using a gray scale detector.

As described above, after the cavities are formed in the supportingmember, a particle may be positioned at the bottom of a cavity using amicromanipulator. This allows the location of a particular particle tobe precisely controlled during the production of the array. The use of amicromanipulator may, however, be impractical for production of sensorarray systems. An alternate method of placing the particles into thecavities may involve the use of a silk screen like process. A series ofmasking materials may be placed on the upper surface of the sensor arrayprior to filling the cavities. The masking materials may be composed ofglass, metal or plastic materials. A collection of particles may beplaced upon the upper surface of the masking materials and the particlesmay be moved across the surface. When a cavity is encountered, aparticle may drop into the cavity if the cavity is unmasked. Thusparticles of known composition are placed in only the unmasked regions.After the unmasked cavities are filled, the masking pattern may bealtered and a second type of particles may be spread across the surface.Preferably, the masking material will mask the cavities that havealready been filled with particle. The masking material may also maskother non-filled cavities. This technique may be repeated until all ofthe cavities are filled. After filling the cavities, a cover may beplaced on the support member, as described above, to inhibit thedisplacement and mixing of the particles. An advantage of such a processis that it may be more amenable to industrial production of supportingmembers.

2. Further System Improvements

One challenge in a chemical sensor system is keeping dead volume to aminimum. This is especially problematic when an interface to the outsideworld is required (e.g., a tubing connection). In many cases the “deadvolume” associated with the delivery of the sample to the reaction sitein a “lab-on-a-chip” may far exceed the actual amount of reagentrequired for the reaction. Filtration is also frequently necessary toprevent small flow channels in the sensor arrays from plugging. Here thefilter can be made an integral part of the sensor package.

In an embodiment, a system for detecting an analyte in a fluid includesa conduit coupled to a sensor array and a vacuum chamber coupled to theconduit. FIG. 38 depicts a system in which a fluid stream (E) passesthrough a conduit (D), onto a sensor array (G), and into a vacuumapparatus (F). The vacuum apparatus (F) may be coupled to the conduit(D) downstream from the sensor array (G). A vacuum apparatus is hereindefined to be any system capable of creating or maintaining a volume ata pressure below atmospheric. Examples of vacuum apparatus includevacuum chambers. Vacuum chamber, in one embodiment, may be sealed tubesfrom which a portion of the air has been evacuated, creating a vacuumwithin the tube. A commonly used example of such a sealed tube is a“vacutainer” system commercially available from Becton Dickinson.Alternatively, a vacuum chamber which is sealed by a movable piston mayalso be used to generate a vacuum. For example, a syringe may be coupledto the conduit. Movement of the piston (i.e., the plunger) away from thechamber will create a partial vacuum within the chamber. Alternatively,the vacuum apparatus may be a vacuum pump or vacuum line. Vacuum pumpsmay include direct drive pumps, oil pumps, aspirator pumps ormicropumps. Micropumps that may be incorporated into a sensor arraysystem have been previously described.

As opposed to previously described methods, in which a pump as used toforce a fluid stream through a sensor array, the use of a vacuumapparatus allows the fluid to be pulled through the sensor array.Referring to FIG. 39, the vacuum apparatus (F) is coupled to downstreamfrom a sensor array. When coupled to the conduit (D), the vacuumapparatus may exert a suction force on the fluid stream, forcing aportion of the stream to pass over, and in some instances, through thesensor array. In some embodiments, the fluid may continue to passthrough the conduit, after passing the sensor array, and into the vacuumapparatus. In an embodiment where the vacuum apparatus is apre-evacuated tube, the fluid flow will continue until the air withinthe tube is at a pressure substantially equivalent to the atmosphericpressure.

The vacuum apparatus may include a penetrable wall (H). The penetrablewall forms a seal inhibiting air from entering the vacuum apparatus.When the wall is broken or punctured, air from outside of the systemwill begin to enter the vacuum apparatus. In one embodiment, the conduitincludes a penetrating member, (e.g., a syringe needle), which allowsthe penetrable wall to be pierced. Piercing the penetrable wall causesair and fluid inside the conduit to be pulled through the conduit intothe vacuum apparatus until the pressure between the vacuum apparatus andthe conduit is equalized.

The sensor array system may also include a filter (B) coupled to theconduit (D) as depicted in FIG. 39. The filter (B) may be positionedalong the conduit, upstream from the sensor array. Filter (B) may be aporous filter which includes a membrane for removing components from thefluid stream. In one embodiment, the filter may include a membrane forremoval of particulates above a minimum size. The size of theparticulates removed will depend on the porosity of the membrane as isknown in the art. Alternatively, the filter may be configured to removeunwanted components of a fluid stream. For example, if the fluid streamis a blood sample, the filter may be configured to remove red and whiteblood cells from the stream, while leaving in the blood stream bloodplasma and other components therein.

The sensor array may also include a reagent delivery reservoir (C). Thereagent delivery system is preferably coupled to the conduit upstreamfrom the sensor array. The reagent delivery reservoir may be formed froma porous material which includes a reagent of interest. As the fluidpasses through this reservoir, a portion of the reagent within theregent delivery reservoir passes into the fluid stream. The fluidreservoir may include a porous polymer or filter paper on which thereagent is stored. Examples of reagents which may be stored within thereagent delivery reservoir include, but are not limited to,visualization agents (e.g., dye or fluorophores), co-factors, buffers,acids, bases, oxidants, and reductants.

The sensor array may also include a fluid sampling device (A) coupled tothe conduit (D). The fluid sampling device is configured to transfer afluid sample from outside the sensor array to the conduit. A number offluid sampling devices may be used including, but not limited to asyringe needle, a tubing connector, a capillary tube, or a syringeadapter.

The sensor array may also include a micropump or a microvalve system,coupled to the conduit to further aid in the transfer of fluid throughthe conduit. Micropumps and valves have been previously described. Inone embodiment, a micro-valve or micropump may be used to keep a fluidsample or a reagent solution separated from the sensor array. Typically,these microvalves and micropumps include a thin flexible diaphragm. Thediaphragm may be moved to an open position, in one embodiment, byapplying a vacuum to the outside of the diaphragm. In this way, a vacuumapparatus coupled to the sensor array may be used to open a remotemicrovalve or pump.

In another embodiment, a microvalve may be used to control theapplication of a vacuum to the system. For example, a microvalve may bepositioned adjacent to the vacuum apparatus. The activation of themicrovalve may allow the vacuum apparatus to communicate with theconduit or sensor array. The microvalve may be remotely activated atcontrolled times and for controlled intervals.

In one embodiment, a sensor array system, such as depicted in FIG. 39,may be used for analysis of blood samples. A micropuncture device (A) isused to extract a small amount of blood from the patient, e.g., througha finger prick. The blood may be drawn through a porous filter thatserves to remove the undesirable particulate matter. For the analysis ofantibodies or antigens in whole blood, the filtering agent may be chosento remove both the white and red blood cells, while leaving in the fluidstream blood plasma and all of the components therein. Methods offiltering blood cells from whole blood are taught, for example, in U.S.Pat. Nos. 5,914,042; 5,876,605, and 5,211,850 which are incorporated byreference. The filtered blood may also be passed through a reagentdelivery reservoir that may consist of a porous layer that isimpregnated with the reagent(s) of interest. In many cases, avisualization agent will be included in this layer so that the presenceof the analytes of interest in the chip can be resolved. The treatedfluid may be passed above the electronic tongue chip through a capillarylayer, down through the various sensing particles and through the chiponto the bottom capillary layer. After exiting the central region, theexcess fluid flows into the vacuum apparatus. This excess fluid mayserve as a source of sample for future measurements should more detailedanalyses be warranted. A “hard copy” of the sample is thus created toback up the electronic data recorded for the specimen.

Other examples of testing procedures for bodily fluids are described inthe following U.S. Pat. Nos. 4,596,657, 4,189,382, 4,115,277, 3,954,623,4,753,776, 4,623,461, 4,069,017, 5,053,197, 5,503,985, 3,696,932,3,701,433, 4,036,946, 5,858,804, 4,050,898, 4,477,575, 4,810,378,5,147,606, 4,246,107, and 4,997,577 all of which are incorporated byreference.

This generally described sampling method may also be used for eitherantibody or antigen testing of bodily fluids. A general scheme for thetesting of antibodies is depicted in FIG. 40. FIG. 40A depicts a polymerbead having a protein coating that can be recognized in a specificmanner by a complimentary antibody. Three antibodies (within the dashedrectangle) are shown to be present in a fluid phase that bathes thepolymer bead. Turning to FIG. 40B, the complimentary antibody binds tothe bead while the other two antibodies remain in the fluid phase. Alarge increase in the complimentary antibody concentration is noted atthis bead. In FIG. 40C a visualization agent such as protein A (withinthe dashed rectangle) is added to the fluid phase. The visualizationagent is chosen because it possesses either a strong absorbance propertyor it exhibits fluorescence characteristics that can be used to identifythe species of interest via optical measurements. Protein A is anexample of a reagent that associates with the common region of mostantibodies. Chemical derivatization of the visualization agent withdyes, quantum particles or fluorophores is used to evoke the desiredoptical characteristics. After binding to the bead-localized antibodies,as depicted in FIG. 40D, the visualization agent reveals the presence ofthe complimentary antibodies at the specific polymer bead sites.

FIG. 41 depicts another general scheme for the detection of antibodieswhich uses a sensor array composed of four individual beads. Each of thefour beads is coated with a different antigen (i.e. a protein coating).As depicted in FIG. 41A, the beads are washed with a fluid sample whichincludes four antibodies. Each of the four antibodies binds to itscomplimentary antigen coating, as depicted in FIG. 41B. A visualizationagent may be introduced into the chamber, as depicted in FIG. 41C. Thevisualization agent, in one embodiment, may bind to the antibodies, asdepicted in FIG. 41D. The presence of the labeled antibodies is assayedby optical means (absorbance, reflectance, fluorescence). Because thelocation of the antigen coatings is known ahead of time, thechemical/biochemical composition of the fluid phase can be determinedfrom the pattern of optical signals recorded at each site.

In an alternative methodology, not depicted, the antibodies in thesample may be exposed to the visualization agent prior to theirintroduction into the chip array. This may render the visualization stepdepicted in 41C unnecessary.

FIG. 42 depicts a system for detecting an analyte in a fluid stream. Thesystem includes a vacuum apparatus, a chamber in which a sensor arraymay be disposed, and an inlet system for introducing the sample into thechamber. In this embodiment, the inlet system is depicted as amicro-puncture device. The chamber holding the sensor array may be aSikes-Moore chamber, as previously described. The vacuum apparatus is astandard “vacutainer” type vacuum tube. The micro puncture deviceincludes a Luer-lock attachment which can receive a syringe needle.Between the micro-puncture device and the chamber a syringe filter maybe placed to filter the sample as the sample enters the chamber.Alternatively, a reagent may be placed within the filter. The reagentmay be carried into the chamber via the fluid as the fluid passesthrough the filter.

As has been previously described, a sensor array may be configured toallow the fluid sample to pass through the sensor array during use. Thefluid delivery to the sensor array may be accomplished by having thefluid enter the top of the chip through the shown capillary (A), asdepicted in FIG. 43. The fluid flow traverses the chip and exits fromthe bottom capillary (B). Between the top and bottom capillaries, thefluid is passed by the bead. Here the fluid containing analytes have anopportunity to encounter the receptor sites. The presence of suchanalytes may be identified using optical means. The light pathway isshown here (D). In the forward flow direction, the beads are typicallyforced towards the bottom of the pit. Under these circumstances, thebead placement is ideal for optical measurements.

In another embodiment, the fluid flow may go from the bottom of thesensor array toward the top of the sensor array, as depicted in FIG. 44.The fluid exits from the top of the chip through the shown capillary(A). The fluid flow traverses the chip and enters from the bottomcapillary (B). Between the top and bottom capillaries, the fluid canavoid the bead somewhat by taking an indirect pathway (C). The presenceof analytes is identified using optical means as before. Unfortunately,only a portion of the light passes through the bead. In the reverse flowdirection, the beads can be dislodged away from the analysis beam by theupwards pressure of the fluid, as shown in FIG. 44. Under thesecircumstances, some of the light may traverse the chip and enter thedetector (not shown) without passing through the sensor bead (Path E).

In any microfluidic chemical sensing system there may be a need to“store” the chemically sensitive elements in an “inert” environment.Typically, the particles may be at least partially surrounded by aninert fluid such as an inert, non reactive gas, a non-reactive solvent,or a liquid buffer solution. Alternatively, the particles may bemaintained under a vacuum. Before exposure of the particles to theanalyte, the inert environment may need to be removed to allow propertesting of the sample. In one embodiment, a system may include a fluidtransfer system for the removal of an inert fluid prior to theintroduction of the sample with minimum dead volume.

In one embodiment, a pumping system may be used to pull the inert fluidthrough from one side (by any pumping action, such as that provided by avacuum downstream from the array). The inert fluid may be efficientlyremoved while the beads remain within the sensor array. Additionally,the analyte sample may be drawn toward the sensor array as the inertfluid is removed from the sensor array. A pocket of air may separate theanalyte sample from the inert fluid as the sample move through theconduit. Alternatively, the sample may be pumped from “upstream” using amicropump. Note that a vacuum downstream can produce a maximum of oneatmosphere of head pressure, while an upstream pump could in principleproduce an arbitrarily high head pressure. This can effect the fluidtransport rates through the system, but for small volume microfluidicsystems, even with low flow coefficients, one atmosphere of headpressure should provide acceptable transfer rates for many applications.

In another embodiment, the vacuum apparatus may be formed directly intoa micromachined array. The vacuum apparatus may be configured totransmit fluid to and from a single cavity or a plurality of cavities.In one embodiment, a separate vacuum apparatus may be coupled to each ofthe cavities.

3. Chemical Improvements

The development of smart sensors capable of discrimination of differentanalytes, toxins, and bacteria has become increasingly important forenvironmental, health and safety, remote sensing, military, and chemicalprocessing applications. Although many sensors capable of highsensitivity and high selectivity detection have been fashioned forsingle analyte detection, only in a few selected cases have arraysensors been prepared which display multi-analyte detectioncapabilities. The obvious advantages of such array systems are theirutility for the analysis of multiple analytes and their ability to be“trained” to respond to new stimuli. Such on site adaptive analysiscapabilities afforded by the array structures makes their utilizationpromising for a variety of future applications.

Single and multiple analyte sensors both typically rely on changes inoptical signals. These sensors typically make use of an indicator thatundergoes a perturbation upon analyte binding. The indicator may be achromophore or a fluorophore. A fluorophore is a molecule that absorbslight at a characteristic wavelength and then re-emits the light mosttypically at a characteristically different wavelength. Fluorophoresinclude, but are not limited to rhodamine and rhodamine derivatives,fluorescein and fluorescein derivatives, coumarins and chelators withthe lanthanide ion series. The emission spectra, absorption spectra andchemical composition of many fluorophores may be found, e.g., in the“Handbook of Fluorescent Probes and Research Chemicals”, R. P. Haugland,ed. which is incorporated herein by reference. A chromophore is amolecule which absorbs light at a characteristic wavelength, but doesnot re-emit light.

As previously described, the receptor itself may incorporate theindicator. The binding of the analyte to the receptor may directly leadto a modulation of the properties of the indicator. Such an approachtypically requires a covalent attachment or strong non-covalent bindingof the indicator onto or as part of the receptor, leading to additionalcovalent architecture. Each and every receptor may need a designedsignaling protocol that is typically unique to that receptor. Generalprotocols for designing in a signal modulation that is versatile andgeneral for most any receptor would be desirable.

In one embodiment, a general method for the creation of optical signalmodulations for most any receptor that is coupled to an immobilizedmatrix has been developed. Immobilized matrices include, but are notlimited to, resins, beads, and polymer surfaces. By immobilization ofthe receptor to the matrix, the receptor is held within a structure thatcan be chemically modified, allowing one to tune and to create anenvironment around the receptor that is sensitive to analyte binding.Coupling of the indicator to an immobilization matrix may make itsensitive to microenvironment changes which foster signal modulation ofthe indicator upon analyte binding. Further, by coupling the indicatorto an immobilization matrix, the matrix itself becomes the signalingunit, not requiring a specific new signaling protocol for each and everyreceptor immobilized on the matrix.

In an embodiment, a receptor for a particular analyte or class ofanalytes may be designed and created with the chemical handlesappropriate for immobilization on and/or in the matrix. A number of suchreceptors have been described above. The receptors can be, but are notlimited to, antibodies, aptamers, organic receptors, combinatoriallibraries, enzymes, and imprinted polymers.

Signaling indicator molecules may be created or purchased which haveappropriate chemical handles for immobilization on and/or in theimmobilization matrix. The indicators may possess chromophores orfluorophores that are sensitive to their microenvironment. Thischromophore or fluorophore may be sensitive to microenvironment changesthat include, but are not limited to, a sensitivity to local pH,solvatophobic or solvatophilic properties, ionic strength, dielectric,ion pairing, and/or hydrogen bonding. Common indicators, dyes, quantumparticles, and semi-conductor particles, are all examples of possibleprobe molecules. The probe molecules may have epitopes similar to theanalyte, so that a strong or weak association of the probe moleculeswith the receptor may occur. Alternatively, the probe molecules may besensitive to a change in their microenvironment that results from one ofthe affects listed in item above.

Binding of the analyte may do one of the following things, resulting ina signal modulation: 1) displace a probe molecule from the binding siteof the receptor, 2) alter the local pH, 3) change the local dielectricproperties, 4) alter the features of the solvent, 5) change thefluorescence quantum yield of individual dyes, 6) alter therate/efficiency of fluorescence resonance energy transfer (FRET) betweendonor-acceptor fluorophore pairs, or 7) change the hydrogen bonding orion pairing near the probe.

In an alternative embodiment, two or more indicators may be attached tothe matrix. Binding between the receptor and analyte causes a change inthe communication between the indicators, again via either displacementof one or more indicators, or changes in the microenvironment around oneor more indicators. The communication between the indicators may be, butis not limited to, fluorescence resonance energy transfer, quenchingphenomenon, and/or direct binding.

In an embodiment, a particle for detecting an analyte may be composed ofa polymeric resin. A receptor and an indicator may be coupled to thepolymeric resin. The indicator and the receptor may be positioned on thepolymeric resin such that the indicator produces a signal in when theanalyte interacts with the receptor. The signal may be a change inabsorbance (for chromophoric indicators) or a change in fluorescence(for fluorophoric indicators).

A variety of receptors may be used, in one embodiment, the receptor maybe a polynucleotide, a peptide, an oligosaccharide, an enzyme, a peptidemimetic, or a synthetic receptor.

In one embodiment, the receptor may be a polynucleotide coupled to apolymeric resin. For the detection of analytes, the polynucleotide maybe a double stranded deoxyribonucleic acid, single strandeddeoxyribonucleic acid, or a ribonucleic acid. Methods for synthesizingand/or attaching a polynucleotide to a polymeric resin are described,for example, in U.S. Pat. No. 5,843,655 which is incorporated herein byreference. “Polynucleotides” are herein defined as chains ofnucleotides. The nucleotides are linked to each other by phosphodiesterbonds. “Deoxyribonucleic acid” is composed of deoxyribonucleotideresidues, while “Ribonucleic acid” is composed of ribonucleotideresidues.

In another embodiment, the receptor may be a peptide coupled to apolymeric resin. “Peptides” are herein defined as chains of amino acidswhose α-carbons are linked through peptide bonds formed by acondensation reaction between the a carboxyl group of one amino acid andthe amino group of another amino acid. Peptides is intended to includeproteins.

Methods for synthesizing and/or attaching a protein or peptides to apolymeric resin are described, for example, in U.S. Pat. Nos. 5,235,028and 5,182,366 which is incorporated herein by reference.

Alternatively, peptide mimetics may be used as the receptor. Peptidesand proteins are sequences of amide linked amino acid building blocks. Avariety of peptide mimetics may be formed by replacing or modifying theamide bond. In one embodiment, the amide bond may be replaced by alkenebonds. In another embodiment, the amide may be replaced by asulphonamide bond. In another embodiment the amino acid sidechain may beplaced on the nitrogen atom, such compounds are commonly known aspeptoids. Peptides may also be formed from non-natural D-stereo-isomersof amino acids. Methods for synthesizing and/or attaching a peptidemimetic to a polymeric resin is described, for example, in U.S. Pat. No.5,965,695 which is incorporated herein by reference.

In another embodiment, the receptor may include an oligosaccharidecoupled to a polymeric resin. An “oligosaccharide” is an oligomercomposed of two or more monosaccharides, typically joined together viaether linkages. Methods for synthesizing and/or attachingoligosaccharides to a polymeric resin are described, for example, inU.S. Pat. Nos. 5,278,303 and 5,616,698 which are incorporated herein byreference.

In another embodiment, polynucleotides, peptides and/or oligosaccharidesmay be coupled to base unit to form a receptor. In one embodiment, thebase unit may have the general structure:

(R¹)_(n)—X—(R²)_(m)

-   -   wherein X comprises carbocyclic systems or C₁-C₁₀ alkanes, n is        an integer of at least 1,    -   m is an integer of at least 1; and    -   wherein each of R¹ independently represents        —(CH₂)_(y)—NR³—C(NR⁴)—NR⁵, —(CH₂)_(y)—NR⁶R⁷, —(CH₂)_(y)—NH—Y,        —(CH₂)_(y)—O-Z;    -   where y is an integer of at least 1;    -   where R³, R⁴, and R⁵ independently represent hydrogen, alkyl,        aryl, alkyl carbonyl of 1 to 10 carbon atoms, or alkoxy carbonyl        of 1 to 10 carbon atoms, or R⁴ and R⁵ together represent a        cycloalkyl group;    -   where R⁶ represents hydrogen, alkyl, aryl, alkyl carbonyl of 1        to 10 carbon atoms, or alkoxy carbonyl of 1 to 10 carbon atoms;    -   where R⁷ represents alkyl, aryl, alkyl carbonyl of 1 to 10        carbon atoms, or alkoxy carbonyl of 1 to 10 carbon atoms;    -   where R⁶ and R⁷ together represent a cycloalkyl group;    -   where Y is a peptide, or hydrogen    -   and where Z is a polynucleotide, an oligosaccharide or hydrogen;        and        wherein each of R² independently represents hydrogen, alkyl,        alkenyl, alkynyl, phenyl, phenylalkyl, arylalkyl, aryl, or        together with another R² group represent a carbocyclic ring. The        use of a base unit such as described above may aid in the        placement and orientation of the side groups to create a more        effective receptor.

The receptor and indicators may be coupled to the polymeric resin by alinker group. A variety of linker groups may be used. The term “linker”,as used herein, refers to a molecule that may be used to link a receptorto an indicator; a receptor to a polymeric resin or another linker, oran indicator to a polymeric resin or another linker. A linker is ahetero or homobifunctional molecule that includes two reactive sitescapable of forming a covalent linkage with a receptor, indicator, otherlinker or polymeric resin. Suitable linkers are well known to those ofskill in the art and include, but are not limited to, straight orbranched-chain carbon linkers, heterocyclic carbon linkers, or peptidelinkers. Particularly preferred linkers are capable of forming covalentbonds to amino groups, carboxyl groups, or sulfhydryl groups or hydroxylgroups. Amino-binding linkers include reactive groups such as carboxylgroups, isocyanates, isothiocyanates, esters, haloalkyls, and the like.Carboxyl-binding linkers are capable of forming include reactive groupssuch as various amines, hydroxyls and the like. Sulfhydryl-bindinglinkers include reactive groups such as sulfhydryl groups, acrylates,isothiocyanates, isocyanates and the like. Hydroxyl binding groupsinclude reactive groups such as carboxyl groups, isocyanates,isothiocyanates, esters, haloalkyls, and the like. The use of some suchlinkers is described in U.S. Pat. No. 6,037,137 which is incorporatedherein by reference.

A number of combinations for the coupling of an indicator and a receptorto a polymeric resin have been devised. These combinations areschematically depicted in FIG. 55. In one embodiment, depicted in FIG.55A, a receptor (R) may be coupled to a polymeric resin. The receptormay be directly formed on the polymeric resin, or be coupled to thepolymeric resin via a linker. An indicator (I) may also be coupled tothe polymeric resin. The indicator may be directly coupled to thepolymeric resin or coupled to the polymeric resin by a linker. In someembodiments, the linker coupling the indicator to the polymeric resin isof sufficient length to allow the indicator to interact with thereceptor in the absence of an analyte.

In another embodiment, depicted in FIG. 55B, a receptor (R) may becoupled to a polymeric resin. The receptor may be directly formed on thepolymeric resin, or be coupled to the polymeric resin via a linker. Anindicator (B) may also be coupled to the polymeric resin. The indicatormay be directly coupled to the polymeric resin or coupled to thepolymeric resin by a linker. In some embodiments, the linker couplingthe indicator to the polymeric resin is of sufficient length to allowthe indicator to interact with the receptor in the absence of ananalyte. An additional indicator (C) may also be coupled to thepolymeric resin. The additional indicator may be directly coupled to thepolymeric resin or coupled to the polymeric resin by a linker. In someembodiments, the additional indicator is coupled to the polymeric resin,such that the additional indicator is proximate the receptor during use.

In another embodiment, depicted in FIG. 55C, a receptor (R) may becoupled to a polymeric resin. The receptor may be directly formed on thepolymeric resin, or be coupled to the polymeric resin via a linker. Anindicator (I) may be coupled to the receptor. The indicator may bedirectly coupled to the receptor or coupled to the receptor by a linker.In some embodiments, the linker coupling the indicator to the polymericresin is of sufficient length to allow the indicator to interact withthe receptor in the absence of an analyte, as depicted in FIG. 55E.

In another embodiment, depicted in FIG. 55D, a receptor (R) may becoupled to a polymeric resin. The receptor may be directly formed on thepolymeric resin, or be coupled to the polymeric resin via a linker. Anindicator (B) may be coupled to the receptor. The indicator may bedirectly coupled to the receptor or coupled to the receptor by a linker.In some embodiments, the linker coupling the indicator to the polymericresin is of sufficient length to allow the indicator to interact withthe receptor in the absence of an analyte, as depicted in FIG. 55F. Anadditional indicator (C) may also be coupled to the receptor. Theadditional indicator may be directly coupled to the receptor or coupledto the receptor by a linker.

In another embodiment, depicted in FIG. 55G, a receptor (R) may becoupled to a polymeric resin. The receptor may be directly formed on thepolymeric resin, or be coupled to the polymeric resin via a linker. Anindicator (B) may be coupled to the polymeric resin. The indicator maybe directly coupled to the polymeric resin or coupled to the polymericresin by a linker. In some embodiments, the linker coupling theindicator to the polymeric resin is of sufficient length to allow theindicator to interact with the receptor in the absence of an analyte. Anadditional indicator (C) may also be coupled to the receptor. Theadditional indicator may be directly coupled to the receptor or coupledto the receptor by a linker.

In another embodiment, depicted in FIG. 55H, a receptor (R) may becoupled to a polymeric resin by a first linker. An indicator (I) may becoupled to the first linker. The indicator may be directly coupled tothe first linker or coupled to the first linker by a second linker. Insome embodiments, the second linker coupling the indicator to thepolymeric resin is of sufficient length to allow the indicator tointeract with the receptor in the absence of an analyte.

In another embodiment, depicted in FIG. 55I, a receptor (R) may becoupled to a polymeric resin by a first linker. An indicator (B) may becoupled to the first linker. The indicator may be directly coupled tothe first linker or coupled to the first linker by a second linker. Insome embodiments, the second linker coupling the indicator to the firstlinker is of sufficient length to allow the indicator to interact withthe receptor in the absence of an analyte. An additional indicator (C)may be coupled to the receptor. The additional indicator may be directlycoupled to the receptor or coupled to the receptor by a linker.

These various combinations of receptors, indicators, linkers andpolymeric resins may be used in a variety of different signallingprotocols. Analyte-receptor interactions may be transduced into signalsthrough one of several mechanisms. In one approach, the receptor sitemay be preloaded with an indicator, which can be displaced in acompetition with analyte ligand. In this case, the resultant signal isobserved as a decrease in a signal produced by the indicator. Thisindicator may be a fluorophore or a chromophore. In the case of afluorophore indicator, the presence of an analyte may be determined by adecrease in the fluorescence of the particle. In the case of achromophore indicator, the presence of an analyte may be determined by adecrease in the absorbance of the particle.

A second approach that has the potential to provide better sensitivityand response kinetics is the use of an indicator as a monomer in thecombinatorial sequences (such as either structure shown in FIG. 14), andto select for receptors in which the indicator functions in the bindingof ligand. Hydrogen bonding or ionic substituents on the indicatorinvolved in analyte binding may have the capacity to change the electrondensity and/or rigidity of the indicator, thereby changing observablespectroscopic properties such as fluorescence quantum yield, maximumexcitation wavelength, maximum emission wavelength, and/or absorbance.This approach may not require the dissociation of a preloadedfluorescent ligand (limited in response time by k_(off)), and maymodulate the signal from essentially zero without analyte to largelevels in the presence of analyte.

In one embodiment, the microenvironment at the surface and interior ofthe resin beads may be conveniently monitored using spectroscopy whensimple pH sensitive dyes or solvachromic dyes are imbedded in the beads.As a guest binds, the local pH and dielectric constants of the beadschange, and the dyes respond in a predictable fashion. The binding oflarge analytes with high charge and hydrophobic surfaces, such as DNA,proteins, and steroids, should induce large changes in localmicroenvironment, thus leading to large and reproducible spectralchanges. This means that most any receptor can be attached to a resinbead that already has a dye attached, and that the bead becomes a sensorfor the particular analyte.

In one embodiment, a receptor that may be covalently coupled to anindicator. The binding of the analyte may perturb the localmicroenvironment around the receptor leading to a modulation of theabsorbance or fluorescence properties of the sensor.

In one embodiment, receptors may be used immediately in a sensing modesimply by attaching the receptors to a bead that is already derivatizedwith a dye sensitive to its microenvironment. This is offers anadvantage over other signalling methods because the signaling protocolbecomes routine and does not have to be engineered; only the receptorsneed to be engineered. The ability to use several different dyes withthe same receptor, and the ability to have more than one dye on eachbead allows flexibility in the design of a sensing particle.

Changes in the local pH, local dielectric, or ionic strength, near afluorophore may result in a signal. A high positive charge in amicroenvironment leads to an increased pH since hydronium migrates awayfrom the positive region. Conversely, local negative charge decreasesthe microenvironment pH. Both changes result in a difference in theprotonation state of pH sensitive indicators present in thatmicroenvironment. Many common chromophores and fluorophores are pHsensitive. The interior of the bead may be acting much like the interiorof a cell, where the indicators should be sensitive to local pH.

The third optical transduction scheme involves fluorescence energytransfer. In this approach, two fluorescent monomers for signaling maybe mixed into a combinatorial split synthesis. Examples of thesemonomers are depicted in FIG. 14. Compound 470 (a derivative offluorescein) contains a common colorimetric/fluorescent probe that maybe mixed into the oligomers as the reagent that will send out amodulated signal upon analyte binding. The modulation may be due toresonance energy transfer to monomer 475 (a derivative of rhodamine).When an analyte binds to the receptor, structural changes in thereceptor will alter the distance between the monomers (schematicallydepicted in FIG. 8, 320 corresponds to monomer 470 and 330 correspondsto monomer 475). It is well known that excitation of fluorescein mayresult in emission from rhodamine when these molecules are orientedcorrectly. The efficiency of resonance energy transfer from fluoresceinto rhodamine will depend strongly upon the presence of analyte binding;thus measurement of rhodamine fluorescence intensity (at a substantiallylonger wavelength than fluorescein fluorescence) will serve as aindicator of analyte binding. To greatly improve the likelihood of amodulatory fluorescein-rhodamine interaction, multiple rhodamine tagscan be attached at different sites along a combinatorial chain withoutsubstantially increasing background rhodamine fluorescence (onlyrhodamine very close to fluorescein will yield appreciable signal). Inone embodiment, depicted in FIG. 8, when no ligand is present, shortwavelength excitation light (blue light) excites the fluorophore 320,which fluoresces (green light). After binding of analyte ligand to thereceptor, a structural change in the receptor molecule bringsfluorophore 320 and fluorophore 330 in proximity, allowing excited-statefluorophore 320 to transfer its energy to fluorophore 330. This process,fluorescence resonance energy transfer, is extremely sensitive to smallchanges in the distance between dye molecules (e.g.,efficiency˜[distance]⁻⁶).

In another embodiment, photoinduced electron transfer (PET) may be usedto analyze the local microenvironment around the receptor. The methodsgenerally includes a fluorescent dye and a fluorescence quencher. Afluorescence quencher is a molecule that absorbs the emitted radiationfrom a fluorescent molecule. The fluorescent dye, in its excited state,will typically absorbs light at a characteristic wavelength and thenre-emit the light at a characteristically different wavelength. Theemitted light, however, may be reduced by electron transfer with thefluorescent quencher, which results in quenching of the fluorescence.Therefore, if the presence of an analyte perturbs the quenchingproperties of the fluorescence quencher, a modulation of the fluorescentdye may be observed.

The above described signalling methods may be incorporated into avariety of receptor-indicator-polymeric resin systems. Turning to FIG.55A, an indicator (I) and receptor (R) may be coupled to a polymericresin. In the absence of an analyte, the indicator may produce a signalin accordance with the local microenvironment. The signal may be anabsorbance at a specific wavelength or a fluorescence. When the receptorinteracts with an analyte, the local microenvironment may be alteredsuch that the produced signal is altered. In one embodiment, depicted inFIG. 55A, the indicator may partially bind to the receptor in theabsence of an analyte. When the analyte is present the indicator may bedisplaced from the receptor by the analyte. The local microenvironmentfor the indicator therefore changes from an environment where theindicator is binding with the receptor, to an environment where theindicator is no longer bound to the receptor. Such a change inenvironment may induce a change in the absorbance or fluorescence of theindicator.

In another embodiment, depicted in Turning to FIG. 55C, an indicator (I)may be coupled to a receptor (R). The receptor may be coupled to apolymeric resin. In the absence of an analyte, the indicator may producea signal in accordance with the local microenvironment. The signal maybe an absorbance at a specific wavelength or a fluorescence. When thereceptor interacts with an analyte, the local microenvironment may bealtered such that the produced signal is altered. In contrast to thecase depicted in FIG. 55A, the change in local microenvironment may bedue to a conformation change of the receptor due to the biding of theanalyte. Such a change in environment may induce a change in theabsorbance or fluorescence of the indicator.

In another embodiment, depicted in FIG. 55E, an indicator (I) may becoupled to a receptor by a linker. The linker may have a sufficientlength to allow the indicator to bind to the receptor in the absence ofan analyte. The receptor (R) may be coupled to a polymeric resin. In theabsence of an analyte, the indicator may produce a signal in accordancewith the local microenvironment. As depicted in FIG. 55E, the indicatormay partially bind to the receptor in the absence of an analyte. Whenthe analyte is present the indicator may be displaced from the receptorby the analyte. The local microenvironment for the indicator thereforechanges from an environment where the indicator is binding with thereceptor, to an environment where the indicator is no longer bound tothe receptor. Such a change in environment may induce a change in theabsorbance or fluorescence of the indicator.

In another embodiment, depicted in FIG. 55H, a receptor (R) may becoupled to a polymeric resin by a first linker. An indicator may becoupled to the first linker. In the absence of an analyte, the indicatormay produce a signal in accordance with the local microenvironment. Thesignal may be an absorbance at a specific wavelength or a fluorescence.When the receptor interacts with an analyte, the local microenvironmentmay be altered such that the produced signal is altered. In oneembodiment, as depicted in FIG. 55H, the indicator may partially bind tothe receptor in the absence of an analyte. When the analyte is presentthe indicator may be displaced from the receptor by the analyte. Thelocal microenvironment for the indicator therefore changes from anenvironment where the indicator is binding with the receptor, to anenvironment where the indicator is no longer bound to the receptor. Sucha change in environment may induce a change in the absorbance orfluorescence of the indicator.

In another embodiment, the use of fluorescence resonance energy transferor photoinduced electron transfer may be used to detect the presence ofan analyte. Both of these methodologies involve the use of twofluorescent molecules. Turning to FIG. 55B, a first fluorescentindicator (B) may be coupled to receptor (R). Receptor (R) may becoupled to a polymeric resin. A second fluorescent indicator (C) mayalso be coupled to the polymeric resin. In the absence of an analyte,the first and second fluorescent indicators may be positioned such thatfluorescence energy transfer may occur. In one embodiment, excitation ofthe first fluorescent indicator may result in emission from the secondfluorescent indicator when these molecules are oriented correctly.Alternatively, either the first or second fluorescent indicator may be afluorescence quencher. When the two indicators are properly aligned, theexcitation of the fluorescent indicators may result in very littleemission due to quenching of the emitted light by the fluorescencequencher. In both cases, the receptor and indicators may be positionedsuch that fluorescent energy transfer may occur in the absence of ananalyte. When the analyte is presence the orientation of the twoindicators may be altered such that the fluorescence energy transferbetween the two indicators is altered. In one embodiment, the presenceof an analyte may cause the indicators to move further apart. This hasan effect of reducing the fluorescent energy transfer. If the twoindicators interact to produce an emission signal in the absence of ananalyte, the presence of the analyte may cause a decrease in theemission signal. Alternatively, if one the indicators is a fluorescencequencher, the presence of an analyte may disrupt the quenching and thefluorescent emission from the other indicator may increase. It should beunderstood that these effects will reverse if the presence of an analytecauses the indicators to move closer to each other.

In another embodiment, depicted in FIG. 55D, a first fluorescentindicator (B) may be coupled to receptor (R). A second fluorescentindicator (C) may also be coupled to the receptor. Receptor (R) may becoupled to a polymeric resin. In the absence of an analyte, the firstand second fluorescent indicators may be positioned such thatfluorescence energy transfer may occur. In one embodiment, excitation ofthe first fluorescent indicator may result in emission from the secondfluorescent indicator when these molecules are oriented correctly.Alternatively, either the first or second fluorescent indicator may be afluorescence quencher. When the two indicators are properly aligned, theexcitation of the fluorescent indicators may result in very littleemission due to quenching of the emitted light by the fluorescencequencher. In both cases, the receptor and indicators may be positionedsuch that fluorescent energy transfer may occur in the absence of ananalyte. When the analyte is presence the orientation of the twoindicators may be altered such that the fluorescence energy transferbetween the two indicators is altered. In one embodiment, depicted inFIG. 55D, the presence of an analyte may cause the indicators to movefurther apart. This has an effect of reducing the fluorescent energytransfer. If the two indicators interact to produce an emission signalin the absence of an analyte, the presence of the analyte may cause adecrease in the emission signal. Alternatively, if one the indicators isa fluorescence quencher, the presence of an analyte may disrupt thequenching and the fluorescent emission from the other indicator mayincrease. It should be understood that these effects will reverse if thepresence of an analyte causes the indicators to move closer to eachother.

In a similar embodiment to FIG. 55D, the first fluorescent indicator (B)and second fluorescent indicator (C) may be both coupled to receptor(R), as depicted in FIG. 55F. Receptor (R) may be coupled to a polymericresin. First fluorescent indicator (B) may be coupled to receptor (R) bya linker group. The linker group may allow the first indicator to bindthe receptor, as depicted in FIG. 55F. In the absence of an analyte, thefirst and second fluorescent indicators may be positioned such thatfluorescence energy transfer may occur. When the analyte is presence,the first indicator may be displaced from the receptor, causing thefluorescence energy transfer between the two indicators to be altered.

In another embodiment, depicted in FIG. 55G, a first fluorescentindicator (B) may be coupled to a polymeric resin. Receptor (R) may alsobe coupled to a polymeric resin. A second fluorescent indicator (C) maybe coupled to the receptor (R). In the absence of an analyte, the firstand second fluorescent indicators may be positioned such thatfluorescence energy transfer may occur. In one embodiment, excitation ofthe first fluorescent indicator may result in emission from the secondfluorescent indicator when these molecules are oriented correctly.Alternatively, either the first or second fluorescent indicator may be afluorescence quencher. When the two indicators are properly aligned, theexcitation of the fluorescent indicators may result in very littleemission due to quenching of the emitted light by the fluorescencequencher. In both cases, the receptor and indicators may be positionedsuch that fluorescent energy transfer may occur in the absence of ananalyte. When the analyte is presence the orientation of the twoindicators may be altered such that the fluorescence energy transferbetween the two indicators is altered. In one embodiment, the presenceof an analyte may cause the indicators to move further apart. This hasan effect of reducing the fluorescent energy transfer. If the twoindicators interact to produce an emission signal in the absence of ananalyte, the presence of the analyte may cause a decrease in theemission signal. Alternatively, if one the indicators is a fluorescencequencher, the presence of an analyte may disrupt the quenching and thefluorescent emission from the other indicator may increase. It should beunderstood that these effects will reverse if the presence of an analytecauses the indicators to move closer to each other.

In another embodiment, depicted in FIG. 55I, a receptor (R) may becoupled to a polymeric resin by a first linker. A first fluorescentindicator (B) may be coupled to the first linker. A second fluorescentindicator (C) may be coupled to the receptor (R). In the absence of ananalyte, the first and second fluorescent indicators may be positionedsuch that fluorescence energy transfer may occur. In one embodiment,excitation of the first fluorescent indicator may result in emissionfrom the second fluorescent indicator when these molecules are orientedcorrectly. Alternatively, either the first or second fluorescentindicator may be a fluorescence quencher. When the two indicators areproperly aligned, the excitation of the fluorescent indicators mayresult in very little emission due to quenching of the emitted light bythe fluorescence quencher. In both cases, the receptor and indicatorsmay be positioned such that fluorescent energy transfer may occur in theabsence of an analyte. When the analyte is presence the orientation ofthe two indicators may be altered such that the fluorescence energytransfer between the two indicators is altered. In one embodiment, thepresence of an analyte may cause the indicators to move further apart.This has an effect of reducing the fluorescent energy transfer. If thetwo indicators interact to produce an emission signal in the absence ofan analyte, the presence of the analyte may cause a decrease in theemission signal. Alternatively, if one the indicators is a fluorescencequencher, the presence of an analyte may disrupt the quenching and thefluorescent emission from the other indicator may increase. It should beunderstood that these effects will reverse if the presence of an analytecauses the indicators to move closer to each other.

In one embodiment, polystyrene/polyethylene glycol resin beads may beused as a polymeric resin since they are highly water permeable, andgive fast response times to penetration by analytes. The beads may beobtained in sizes ranging from 5 microns to 250 microns. Analysis with aconfocal microscope reveals that these beads are segregated intopolystyrene and polyethylene glycol microdomains, at about a 1 to 1ratio. Using the volume of the beads and the reported loading of 300pmol/bead we can calculate an average distance of 35 Å between terminalsites. This distance is well within the Forester radii for thefluorescent dyes that we are proposing to use in our fluorescenceresonance energy transfer (“FRET”) based signaling approaches. Thisdistance is also reasonable for communication between binding events andmicroenvironment changes around the fluorophores.

The derivatization of the beads with our receptors and with theindicators may be accomplished by coupling carboxylic acids and aminesusing EDC and HOBT. Typically, the efficiency of couplings are greaterthat 90% using quantitative ninhydrin tests. (See Niikura, K.; Metzger,A.; and Anslyn, E. V. “A Sensing Ensemble with Selectivity for IositolTrisphosphate”, J. Am. Chem. Soc. 1998, 120, 0000, which is incorporatedherein by reference). The level of derivatization of the beads issufficient to allow the loading of a high enough level of indicators andreceptors to yield successful assays. However, an even higher level ofloading may be advantageous since it would increase the multi-valencyeffect for binding analytes within the interior of the beads. We mayincrease the loading level two fold and ensure that two amines are closein proximity by attaching an equivalent of lysine to the beads (see FIG.45D). The amines may be kept in proximity so that binding of an analyteto the receptor will influence the environment of a proximal indicator.

Even though a completely random attachment of indicator and a receptorlead to an effective sensing particle, it may be better to rationallyplace the indicator and receptor in proximity. In one embodiment, lysinethat has different protecting groups on the two different amines may beused, allowing the sequential attachment of an indicator and a receptor.If needed, additional rounds of derivatization of the beads with lysinemay increase the loading by powers of two, similar to the synthesis ofthe first few generations of dendrimers.

In contrast, too high a loading of fluorophores will lead toself-quenching, and the emission signals may actually decrease withhigher loadings. If self quenching occurs for fluorophores on thecommercially available beads, we may incrementally cap the terminalamines thereby giving incrementally lower loading of the indicators.

Moreover, there should be an optimum ratio of receptors to indicators.The optimum ratio is defined as the ratio of indicator to receptor togive the highest response level. Too few indicators compared toreceptors may lead to little change in spectroscopy since there will bemany receptors that are not in proximity to indicators. Too manyindicators relative to receptors may also lead to little change inspectroscopy since many of the indicators will not be near receptors,and hence a large number of the indicators will not experience a changein microenvironment. Through iterative testing, the optimum ratio may bedetermined for any receptor indicator system.

This iterative sequence will be discussed in detail for a particledesigned to signal the presence of an analyte in a fluid. The sequencebegins with the synthesis of several beads with different loadings ofthe receptor. The loading of any receptor may be quantitated using theninhydrin test. (The ninhydrin test is described in detail in Kaiser,E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. “Color Test forDetection of Free Terminal Amino Groups in the Solid-Phase Synthesis ofPeptides”, Anal. Biochem. 1970, 34, 595-598 which is incorporated hereinby reference). The number of free amines on the bead is measured priorto and after derivatization with the receptor, the difference of whichgives the loading. Next, the beads undergo a similar analysis withvarying levels of molecular probes. The indicator loading may bequantitated by taking the absorption spectra of the beads. In thismanner, the absolute loading level and the ratio between the receptorand indicators may be adjusted. Creating calibration curves for theanalyte using the different beads will allow the optimum ratios to bedetermined.

The indicator loading may be quantitated by taking the absorptionspectra of a monolayer of the beads using our sandwich technique (SeeFIG. 46D). The sandwich technique involves measuring the spectroscopy ofsingle monolayers of the beads. The beads may be sandwiched between twocover slips and gently rubbed together until a monolayer of the beads isformed. One cover slip is removed, and mesh with dimensions on the orderof the beads is then place over the beads, and the cover slip replaced.This sandwich is then placed within a cuvette, and the absorbance oremission spectra are recorded. Alternatively, an sensor array system, asdescribed above, may be used to analyze the interaction of the beadswith the analyte.

A variety of receptors may be coupled to the polymeric beads. Many ofthese receptors have been previously described. Other receptors areshown in FIG. 47.

As described generally above, an ensemble may be formed by a syntheticreceptor and a probe molecule, either mixed together in solution orbound together on a resin bead. The modulation of the spectroscopicproperties of the probe molecule results from perturbation of themicroenvironment of the probe due to interaction of the receptor withthe analyte; often a simple pH effect. The use of a probe moleculecoupled to a common polymeric support may produce systems that givecolor changes upon analyte binding. A large number of dyes arecommercially available, many of which may be attached to the bead via asimple EDC/HOBT coupling (FIG. 48 shows some examples of indicators).These indicators are sensitive to pH, and also respond to ionic strengthand solvent properties. When contacted with an analyte, the receptorinteracts with the analyte such that microenvironment of the polymericresin may become significantly changed. This change in themicroenvironment may induce a color change in the probe molecule. Thismay lead to an overall change in the appearance of the particleindicating the presence of the analyte.

Since many indicators are sensitive to pH and local ionic strength,index of refraction, and/or metal binding, lowering the local dielectricconstant near the indicators may modulate the activity of the indicatorssuch that they are more responsive. A high positive charge in amicroenvironment leads to an increased pH since hydronium ions migrateaway from the positive region. Conversely, local negative chargedecreases the microenvironment pH. Both changes result in a differenceon the protonation state of a pH sensitive indicator present in thatmicroenvironment. The altering of the local dielectric environment maybe produced by attaching molecules of differing dielectric constants tothe bead proximate to the probe molecules. Examples of molecules whichmay be used to alter the local dielectric environment include, but arenot limited to, planar aromatics, long chain fatty acids, and oligomerictracts of phenylalanine, tyrosine, and tryptophan. Differing percentagesof these compounds may be attached to the polymeric bead to alter thelocal dielectric constant.

Competition assays may also be used to produce a signal to indicate thepresence of an analyte. The high specificity of antibodies makes themthe current tools of choice for the sensing and quantitation ofstructurally complex molecules in a mixture of analytes. These assaysrely on a competition approach in which the analyte is tagged and boundto the antibody. Addition of the untagged analyte results in a releaseof the tagged analytes and spectroscopic modulation is monitored.Surprisingly, although competition assays have been routinely used todetermine binding constants with synthetic receptors, very little workhas been done exploiting competition methods for the development ofsensors based upon synthetic receptors. Yet, all the ways in which themicroenvironment of the chromophore can be altered, as described above,may be amenable to the competition approach. Those that have beendeveloped using synthetic receptors are mostly centered upon the use ofcyclodextrins. (See e.g., Hamasaki, K.; Ikeda, H.; Nakamura, A.; Ueno,A.; Toda, F.; Suzuki, I.; Osa, T. “Fluorescent Sensors of MolecularRecognition. Modified Cyclodextrins Capable of ExhibitingGuest-Responsive Twisted Intramolecular Charge Transfer Fluorescence” J.Am. Chem. Soc. 1993, 115, 5035, and reference (5) therein, which areincorporated herein by reference) A series of parent and derivatizedcyclodextrins have been combined with chromophores that are responsiveto the hydrophobicity of their microenvironment to produce a sensorsystem. Displacement of the chromophores from the cyclodextrin cavity bybinding of a guest leads to a diagnostic spectroscopy change.

This competitive approach has been used successfully, in one embodiment,for the detection of carbohydrates such as inositol-1,4,5-triphosphate(IP₃). In one embodiment, a synthetic receptor 5 may be paired with anoptical signaling molecule 5-carboxyfluorescein, to quantitate IP₃ at nMconcentrations. A competition assay employing an ensemble of5-carboxyfluorescein and receptor 5 was used to measure bindingconstants. The addition of receptor 5 to 5-carboxyfluorescein resultedin a red shift of the absorption of 5-carboxyfluorescein. Monitoring theabsorption at 502 nm, followed by analysis of the data using theBenesi-Hildebrand method, gave affinity constants of 2.2×10⁴ M⁻¹ for5-carboxyfluorescein binding to receptor 5. Addition of IP₃ to asolution of the complexes formed between 5 and 5-carboxyfluoresceinresulted in displacement of 5-carboxyfluorescein and a subsequent blueshift.

In order to enhance the affinity of receptor 5 for IP₃, similar assayswere repeated in methanol, and with 2% of the surfactant Triton-X. Inmethanol and the detergent solutions, 5-carboxyfluorescein prefers acyclized form in which the 2-carboxylate has undergone an intramolecularconjugate addition to the quinoid structure. This form of5-carboxyfluorescein is colorless and nonfluorescent. Upon addition ofreceptor 5 the yellow color reappears as does the fluorescence. Thepositive character of the receptor induces a ring opening to give thecolored/fluorescent form of 5-carboxyfluorescein. Using theBenesi-Hildebrand method applied to absorption data a binding constantof 1.2×10⁵ M⁻¹ was found for receptor 5 and 5-carboxyfluorescein. Asanticipated based upon the differences in the spectroscopy of5-carboxyfluorescein when it is bound to receptor 5 or free in solution,addition of IP₃ to a solution of receptor 5 and 5-carboxyfluoresceinresulted in a decrease of absorbance and fluorescence due to release of5-carboxyfluorescein into the methanol solution. Binding constants of1.0×10⁸ M⁻¹ and 1.2×10⁷ M⁻¹ for IP₃ and receptor 5 were found formethanol and the surfactant solution respectively.

Since fluorescence spectroscopy is a much more sensitive technique thanUV/visible spectroscopy, and the use of methanol gave significantlystronger binding between receptor 5 and 5-carboxyfluorescein, as well asbetween receptor 5 and IP₃, the monitoring of fluorescence was found tobe the method of choice for sensing nM concentrations of IP₃. We findthat the addition of IP₃ to an ensemble of receptor 5 and5-carboxyfluorescein in water may detect and quantitate IP₃ at aconcentration as low as 1 mM. Importantly, in methanol a 10 nM IP₃concentration was easily detected. A detection level in the nM range isappropriate for the development of an assay using methanol or surfactantas an eluent and capillary electrophoresis to sample and fractionatecellular components.

We have shown that receptor 5 binds IP₃ quite selectively over othersimilarly charged species present in cells. Polyanions with chargeshigher than IP₃, such as IP₄, IP₅, and oligonucleotides, however, areexpected to bind with higher affinities. In order to fractionate thecellular components during signal transduction, and specifically monitorIP₃, a combination of a chemically sensitive particle and capillaryelectrophoresis (CE) may be used. As has been described above, a sensorarray may include a well in which the particle is placed, along with agroove in which the capillary will reside. The capillary will terminatedirectly into the interior of the bead (See FIG. 49). Illumination fromabove and CCD analysis from below may be used to analyze the particle.Samples as small as 100 femtoliters may be introduced into anelectrophoresis capillary for analysis. Using high sensitivitymultiphoton-excited fluorescence as few as ˜50,000 molecules of variousprecursors/metabolites of the neurotransmitter, serotonin may bedetected. Cytosolic samples may be collected and fractionated inmicron-diameter capillary electrophoresis channels. At the capillaryoutlet, components may migrate from the channel individually, and willbe directed onto a bead that houses immobilized receptor 5 and the dyesappropriate for our various signaling strategies. Receptor binding ofIP₃ or IP₄ will elicit modulations in the emission and/or absorptionproperties.

Dramatic spectroscopy changes accompany the chelation of metals toligands that have chromophores. In fact, most colorimetric/fluorescentsensors for metals rely upon such a strategy. Binding of the metal tothe inner sphere of the ligand leads to ligand/metal charge transferbands in the absorbance spectra, and changes in the HOMO-LUMO gap thatleads to fluorescence modulations.

In one embodiment, the binding of an analyte may be coupled with thebinding of a metal to a chromophoric ligand, such that the metal may beused to trigger the response of the sensor for the analyte. The compoundknown as Indo-1 (see FIG. 50 for the structure and emission properties)is a highly fluorescent indicator that undergoes a large wavelengthshift upon exposure to Ca(II). Further, compound 2 binds Ce(III) and theresulting complex is fluorescent. In one embodiment, the binding ofCa(II) or Ce(III) to these sensors may be altered by the addition of ananalyte of interest. By altering the binding of these metals to areceptor a signal may be generated indicating the presence of thereceptor.

In one embodiment, fluorescent indicators that have been used to monitorCa(II) and Ce(II) levels in other applications may be applied to apolymeric supported system. Using the Ca(II) sensor Indo-1 as anexample, the strategy is shown in FIG. 51. Indo-1 binds Ca(II) at nMconcentrations (see FIG. 50). Attachment of Indo-1 and one of ourguanidinium/amine based receptors 3-6 to a resin bead (derivatized withlysine as depicted in FIG. 45D) may lead to intramolecular interactionsbetween the carboxylates of Indo-1 and the guanidiniums/ammoniums of areceptor. The coordination of the carboxylates of Indo-1 may result in adecreased affinity for Ca(II). However, there should be cooperativebinding of Ca(II) and our analytes. Once one of the anionic analytes isbound to its respective receptor, it will competitively displace thecarboxylates of Indo-1 leading to increased Ca(II) binding, which inturn will result in a fluorescence modulation. Similarly, binding ofCa(II) to Indo-1 leaves the guanidiniums of the receptors free to bindcitrate. The assays will likely be most sensitive at concentrations ofthe analytes and Ca(II) near their dissociation constants, where neitherreceptor is saturated and small changes in the extent of binding lead tolarge changes in fluorescence.

We also may switch the role of the metal and the ligand. Indo-1 isfluorescent with and without the Ca(II). However, compound 2 is notfluorescent until Ce(III) binds to it. Thus, a similar assay that reliesupon a change of microenvironment in the interior of the bead dependingupon the presence or absence of the analyte should perturb the bindingof Ce(III) to compound 2. In this case, a repulsive interaction ispredicted for the binding of Ce(III) when the positive charges of theguanidinium based receptors are not neutralized by binding to theanionic analytes.

In one embodiment, an indicator may be coupled to a bead and further maybe bound to a receptor that is also coupled to the bead. Displacement ofthe indicator by an analyte will lead to signal modulation. Such asystem may also take advantage of fluorescent resonance energy transferto produce a signal in the presence of an analyte. Fluorescenceresonance energy transfer is a technique that can be used to shift thewavelength of emission from one position to another in a fluorescencespectra. In this manner it creates a much more sensitive assay since onecan monitor intensity at two wavelengths. The method involves theradiationless transfer of excitation energy from one fluorophore toanother. The transfer occurs via coupling of the oscillating dipoles ofthe donor with the transition dipole of the acceptor. The efficiency ofthe transfer is described by equations first derived by Forester. Theyinvolve a distance factor (R), orientation factor (k), solvent index ofrefraction (N), and spectral overlap (J).

In order to incorporate fluorescence resonance energy transfer into aparticle a receptor and two different indicators may be incorporatedonto a polymeric bead. In the absence of an analyte the fluorescenceresonance energy transfer may occur giving rise to a detectable signal.When an analyte interacts with a receptor, the spacing between theindicators may be altered. Altering this spacing may cause a change inthe fluorescence resonance energy transfer, and thus, a change in theintensity or wavelength of the signal produced. The fluorescenceresonance energy transfer efficiency is proportional to the distance (R)between the two indicators by 1/R⁶. Thus slight changes in the distancebetween the two indicators may induce significant changes in thefluorescence resonance energy transfer.

In one embodiment, various levels of coumarin and fluorescein may beloaded onto resin beads so as to achieve gradiations in FRET levels fromzero to 100%. FIG. 52 shows a 70/30 ratio of emission from5-carboxyfluorescein and coumarin upon excitation of coumarin only inwater. However, other solvents give dramatically different extents ofFRET. This shows that the changes in the interior of the beads does leadto a spectroscopic response. This data also shows that differentialassociation of the various solvents and 5-carboxyfluorescein on resinbeads as a function of solvents. This behavior is evoked from thesolvent association with the polymer itself, in the absence ofpurposefully added receptors. We may also add receptors which exhibitstrong/selective association with strategic analytes. Such receptors mayinduce a modulation in the ratio of FRET upon analyte binding, withinthe microenvironment of the polystyrene/polyethylene glycol matrices.

In order to incorporate a wavelength shift into a fluorescence assays,receptors 3-6 may be coupled to the coumarin/5-carboxyfluorescein beadsdiscussed above. When 5-carboxyfluorescein is bound to the variousreceptors and coumarin is excited, the emission will be primarily formcoumarin since the fluorescein will be bound to the receptors. Upondisplacement of the 5-carboxyfluorescein by the analytes, emissionshould shift more toward 5-carboxyfluorescein since it will be releasedto the bead environment which possesses coumarin. This will give us awavelength shift in the fluorescence which is inherently more sensitivethan the modulation of intensity at a signal wavelength.

There should be large changes in the distance between indicators (R) onthe resin beads. When the 5-carboxyfluorescein is bound, thedonor/acceptor pair should be farther than when displacement takesplace; the FRET efficiency scales as 1/R⁶. The coumarin may be coupledto the beads via a floppy linker, allowing it to adopt manyconformations with respect to a bound 5-carboxyfluorescein. Hence, it ishighly unlikely that the transition dipoles of the donor and acceptorwill be rigorously orthogonal.

In one embodiment, a receptor for polycarboxylic acids and anappropriate probe molecule may be coupled to a polymeric resin to form aparticle for the detection of polycarboxylic acid molecules. Receptorsfor polycarboxylic acids, as well as methods for their use in thedetection of polycarboxylic acids, have been described in U.S. Pat. No.6,045,579 which is incorporated herein by reference. This systeminvolves, in one embodiment, the use of a receptor 3 which was found tobe selective for the recognition of a tricarboxylic acid (e.g., citrate)in water over dicarboxylates, monocarboxylates, phosphates, sugars, andsimple salts. The receptor includes guanidinium groups for hydrogenbonding and charge pairing with the tricarboxylic acid.

An assay for citrate has employed an ensemble of 5-carboxyfluoresceinand 3. The binding between 3 and 5-carboxyfluorescein resulted in alowering of the phenol pK_(a) of 5-carboxyfluorescein, due to thepositive microenvironment presented by 3. This shift in pK_(a) (localpH) caused the phenol moiety to be in a higher state of protonation when5-carboxyfluorescein was free in solution. The absorbance orfluorescence of 5-carboxyfluorescein decreases with higher protonationof the phenol. The intensity of absorbance increases with addition ofhost 3 to 5-carboxyfluorescein, and as predicted the intensity decreasesupon addition of citrate to the ensemble of 3 and 5-carboxyfluorescein.The same effect was seen in the fluorescence spectrum (λmax=525 nm).

In an embodiment, a metal may be used to trigger the response of achromophore to the presence of an analyte. For example, compound 7 bindsCu(II) with a binding constant of 4.9×10⁵ M⁻¹ (See FIG. 53). Addition of1 eq. of Cu(II) increases the binding constant of citrate to compound 7by a factor of at least 5. Importantly, the addition of citrateincreases the binding of Cu(II) to the receptor by a factor of at least10. Therefore the citrate and Cu(II) enhance each other's binding in acooperative manner. Further, the emission spectra of compound 7 is quitesensitive to the addition of citrate when Cu(II) is present, but has noresponse to the addition of citrate in the absence of Cu(II). Thus thebinding of a “trigger” may be perturbed with an analyte of interest, andthe perturbation of the binding of the trigger may be used tospectroscopically monitor the binding of the analyte. The triggering ofthe sensing event by an added entity is similar to the requirement forenzymes in saliva to degrade food particulants into tastantsrecognizable by the receptors on mammalian taste buds.

In one embodiment, citrate receptor 3 may be immobilized on apolystyrene/polyethylene glycol bead, where on the same bead may also beattached a fluorescent probe molecule (FIG. 54). Solutions of citrate atdifferent concentrations may be added to the beads, and the fluorescencespectra of the monolayer recorded. We find exactly the same fluorescenceresponse toward citrate for the ensemble of receptor 3 and5-carboxyfluorescein on the beads as in solution. Apparently, a similarmicroenvironment change to modulate the spectroscopy of5-carboxyfluorescein occurs in the beads, although both5-carboxyfluorescein and receptor 3 are just randomly placed throughoutthe bead.

Additional sensor system include sensors for tartrate and tetracyclin.Compound 4 binds tartrate in buffered water (pH 7.4) with a bindingconstant of approximately 10⁵ M⁻¹. The binding is slow on the NMR timescale, since we can observe both the bound and free receptor andtartrate. This binding is surprisingly strong for pure water. It mustreflect good cooperativity between the host's boronic acid moiety andthe two guanidinium groups for the recognition of the guest's vicinaldiol and two carboxylates respectively. Compound 6 may act as amolecular receptor for tetracyclin. The compound has been synthesized,and by variable temperature NMR it has been found to be in a bowlconformation. Its binding properties with several indicators have beenexplored (most bind with affinities near 10⁴ M⁻¹). More importantly, thebinding of tetracyclin has also been explored, and our preliminaryresults suggests that the binding constant in water is above 10³ M⁻¹.

In another embodiment, a sensing particle may include an oligomer ofamino acids with positively charged side chains such as the lysinetrimer, depicted in FIG. 56, designed to act as the anion receptor, andan attached FRET pair for signaling. Sensing of different anions may beaccomplished by optically monitoring intensity changes in the signal ofthe FRET pair as the analyte interacts with the oligomer.

Upon introduction of an anionic species to 1, the analyte may bind tothe trimer, disturbing the trimer-fluorescein interaction, thereby,altering the fluorescein's ability to participate in the energy transfermechanism. Using a monolayer of resin in a conventional fluorometer, theratio of D:A emission for the FRET pair attached to TG-NH₂ resin issensitive to different solvents as well as to the ionic strength of thesolution. Epifluorescence studies may be performed to test the solventdependence, ionic strength, and binding effects of different anions onthe FRET TG-NH₂ resins. The images of the FRET TG-NH₂ resins within asensor array, taken by a charged coupled device (CCD) may result inthree output channels of red, green, and blue light intensities. The RGBlight intensities will allow for comparison of the results obtainedusing a conventional fluorometer.

The signal transduction of 1 may be studied using a standard fluorometerand within the array platform using epifluorescence microscopy. The RGBanalysis may be used to characterize the relative changes in emission ofthe FRET pair. Other resin-bound sensors may be synthesized by varyingthe amino acid subunits within the oligomers and the length of thepeptide chains.

In another embodiment, solvatochromic dyes may be covalently linked to areceptor unit tethered to a resin bead that is capable of binding tosmall organic guests. In one example, dansyl and dapoxyl may act assensitive probes of their microenvironment. When selecting a dye foruse, characteristics such as high extinction coefficients, highfluorescence quantum yields, and large Stoke's shifts should beconsidered. Dapoxyl and dansyl were anchored to 6% agarose resin beads,in an effort to enhance the signaling response of these resin boundfluorophores in various solvent systems. Agarose beads are crosslinkedgalactose polymers that are more hydrophilic than thepolystyrene-polyethylene glycol resins. The attachment of thesesolvatochromic dyes to the agarose resin beads is outlined in FIG. 57.

The dapoxyl labeled resin (6) was formed by reductively aminatingglyoxalated agarose resin with mono (Fmoc)-butyldiamine hydrochloridesalt using sodium borohydride as the reducing agent. The base labileprotecting group, Fmoc, was removed from 3 with dilute base, and thesolvatochromic dye was anchored to 4 through a reaction to form asulfonamide bond resulting in 6. The tethering of dansyl to agaroseresin was performed similarly.

Analysis of the agarose resins derivatized with dansyl and dapoxyl wasattempted several times using a monolayer sample cell in a conventionalfluorometer. However, satisfactory emission spectra of 5 and 6 indifferent solvent systems were not obtained due to the fragile nature ofthe agarose resin which placed restrictions on the manufacturing of themonolayer sample cell.

Significant signal enhancement of 5 and 6 was seen when the solventsystem was changed from a 50 mM phosphate buffer (pH=7.0) to ethanol(EtOH), methanol (MeOH), and acetonitrile (CH₃CN). The emission of 6increased three fold in EtOH and five fold in CH₃CN when compared to theemission of 6 in a buffer. The agarose-dansyl resin, 5, demonstratedsimilar trends in response to different solvents; however, theintensities were smaller than for 6. For instance, the emission of 5 inEtOH for the red channel was 61% smaller in intensity units compared to6 (2200 vs. 5800 arbitrary intensity units). This observation has beenattributed to the lower quantum yield of fluorescence and the smallerextinction coefficient of dansyl to that of dapoxyl. From these initialstudies, the average fluorescence intensity of the three beads of type 6in EtOH across the red channel was 5800±300 arbitrary intensity countswith a percent standard deviation of 5.0%. Also, before changing to anew solvent, the agarose beads were flushed with the buffer for 5minutes in order to return the agarose-dye resin to a “zero” point ofreference. The background variance of the fluorescence intensity of 6when exposed to each of the buffer washes between each solvent systemwas 5.0% and 4.0% in the red and green channels, respectively.

The response of 5 and 6 to varying ratios of two different solvents wasalso studied. As seen in FIG. 58, a detectable decrease in the emissionof 6 is observed as the percent of the 50 mM phosphate buffer (pH=7) isincreased in ethanol. The fluorescence intensity of 6 decreased by threefold from its original value in 100% EtOH to 100% buffer. There was anincremental decrease in the fluorescence emission intensities of 6 inboth the red and green channels. Once again, 5 demonstrated similartrends in response to the varying ratios of mixed solvent systems;however, the intensities were smaller than 6.

In another example, each dye was derivatized with benzyl amine (2-4) forstudies in solution phase and anchored to resin (5-7) for studies usingthe sandwich method and epi-fluorescence. The dyes and correspondingresins are depicted in FIG. 59.

Fluorescence studies have been performed for each dye in solution phaseand attached to resin. FIG. 60 illustrates an example of the emissionchanges in 4 (part A.) and 7 (part B.) that result from exposure todifferent solvent systems. The quantum yield of 4 diminished in morepolar protic media (i.e. ethanol); whereas, the quantum yield of 4increased in more hydrophobic environments (i.e. cyclohexane). Also, theStoke's shift of each probe changed significantly between nonpolar andpolar media. For example, the Stoke's shift of 4 (λ_(em)-λ_(abs)) in 1:1mixture of methanol and 1.0 M aqueous phosphate buffer was 221 nm, butthe Stoke's shift of 4 was 80 nm in cyclohexane. 7 displayed similartrends, but the Stoke's shift from solvent to solvent was not asdramatic. The optical properties of 5-7 only varied slightly whencompared to their homogeneous analogs.

Of the three fluorophores, the solvatochromic properties of coumarinwere not as dramatic when compared to dansyl and dapoxyl. 6 and 7displayed the largest Stoke's shifts. The emission wavelength for 5-7red shifted when placed in more polar solvents. However, when 6 wasplaced in water, the Stoke's shift was the same as in when placed incyclohexane as seen in FIG. 60. This trend was observed with eachfluoresently labeled resin, and may be explained by the fact that theseprobes are hydrophobic and that they may actually reside within thehydrophobic core of the PEG-PS resin when submerged in water.

In another example a selective chemosensor for ATP was found. A beadwith a polyethylene-glycol base was attached via guanidinium to two longpolypeptide arms that were known to interact with the adenine group ofATP, as depicted in FIG. 61. The tripeptide arms contained twofluorophore attachment sites for 5-carboxyfluorescein (fluorescein), andan attachment site for 7-diethylaminocoumarin-3-carboxylic acid(coumarin) located on the terminal end of the lysine that was attachedto the core structure. The fluorophores act as receptors for the desiredanalyte. The fluorophores also act as indicators to signal changes inthe environment before and after the addition of analytes.

Fluorescently labeled N-methylanthraniloyl-ATP were chosen to screen forATP receptors. Sequences of amino acids were linked as tripeptides andequilibrated with a buffer. The resin was transferred to a microscopeslide and illuminated with UV light. The results yielded 6 sequenceswith active beads that displayed fluorescent activity, and 3 sequenceswith inactive beads where there was no detectable fluorescent activity.

Three of the 6 active beads, and 1 of the 3 inactive beads werearbitrarily chosen to react with ATP (Sequences below in bold). When thefluorescein and coumarin were excited there was no detectable differencein the FRET upon addition of ATP. This may be due to there being anaverage distance between the fluorophores within the beads which doesnot significantly change upon binding ATP. However, all but one activebead (Thr-Val-Asp) exhibited a fluorescence modulation upon excitationof fluorescein. The lack of response from an active bead shows thatscreening against a derivatized analyte (MANT-ATP in this case) will notguarantee that the active beads are successful sensors when synthesizedwith attached fluorophores. Either this active bead binds the MANTprotion of MANT-ATP or there is no significant microenvironment changearound the fluorophores of the Thr-Val-Asp receptor upon binding ATP.

Active Beads Inactive Beads His-Ala-Asp His-Phe-Gly Glu-Pro-ThrSer-Ala-Asp Thr-Val-Asp Trp-Asn-Glu Met-Thr-His Asp-Ala-Asp Ser-Tyr-Ser

A large spectral response upon addition of ATP was observed with theSer-Tyr-Ser sequence in the active bead. The increase in fluoresceinemission is possibly due to a higher local pH around the fluoresceinupon binding of ATP. Further studies were performed with the Ser-Tyr-Sersequence and analytes, AMP, and GTP, which are structurally similar toATP. This peptidic library member exhibited very high detectionselectivity for ATP over these structurally similar potentiallycompeting analytes. The lack of response to AMP suggests the necessityfor triphosphates to bind strongly to the guanidinium entities of thereceptor, while the lack of response to GTP indicates the specificityfor nucleotide bases imparted by the tripeptide arms. The combination ofserine and tyrosine suggests π-stacking between the phenol of tyr andadenine and hydrogen bonding interactions between the serine OH and/orthe ribose or adenine. These studies have demonstrated that the union ofa proven core with combinatorial methods, followed by the attachment offluorophores, can create resin bound chemosensors with excellentselectivity.

As described above, a particle, in some embodiments, possesses both theability to interact with the analyte of interest and to create amodulated signal. In one embodiment, the particle may include receptormolecules which undergo a chemical change in the presence of the analyteof interest. This chemical change may cause a modulation in the signalproduced by the particle. Chemical changes may include chemicalreactions between the analyte and the receptor. Receptors may includebiopolymers or organic molecules. Such chemical reactions may include,but are not limited to, cleavage reactions, oxidations, reductions,addition reactions, substitution reactions, elimination reactions, andradical reactions.

In one embodiment, the mode of action of the analyte on specificbiopolymers may be taken advantage of to produce an analyte detectionsystem. As used herein biopolymers refers to natural and unnatural:peptides, proteins, polynucleotides, and oligosaccharides. In someinstances, analytes, such as toxins and enzymes, will react withbiopolymer such that cleavage of the biopolymer occurs. In oneembodiment, this cleavage of the biopolymer may be used to produce adetectable signal. A particle may include a biopolymer and an indicatorcoupled to the biopolymer. In the presence of the analyte the biopolymermay be cleaved such that the portion of the biopolymer which includesthe indicator may be cleaved from the particle. The signal produced fromthe indicator is then displaced from the particle. The signal of thebead will therefore change thus indicating the presence of a specificanalyte.

Proteases represent a number of families of proteolytic enzymes thatcatalytically hydrolyze peptide bonds. Principal groups of proteasesinclude metalloproteases, serine porteases, cysteine proteases andaspartic proteases. Proteases, in particular serine proteases, areinvolved in a number of physiological processes such as bloodcoagulation, fertilization, inflammation, hormone production, the immuneresponse and fibrinolysis.

Numerous disease states are caused by and may be characterized byalterations in the activity of specific proteases and their inhibitors.For example emphysema, arthritis, thrombosis, cancer metastasis and someforms of hemophilia result from the lack of regulation of serineprotease activities. In case of viral infection, the presence of viralproteases have been identified in infected cells. Such viral proteasesinclude, for example, HIV protease associated with AIDS and NS3 proteaseassociated with Hepatitis C. Proteases have also been implicated incancer metastasis. For example, the increased presence of the proteaseurokinase has been correlated with an increased ability to metastasizein many cancers.

In one embodiment, the presence of a protease may be detected by the useof a biopolymer coupled to a polymeric resin. For the detection ofproteases, the biopolymer may be a protein or peptide. Methods forsynthesizing and/or attaching a protein or peptides to a polymeric resinare described, for example, in U.S. Pat. No. 5,235,028 which isincorporated herein by reference. “Proteins” and “peptides” are hereindefined as chains of amino acids whose a-carbons are linked throughpeptide bonds formed by a condensation reaction between the a carboxylgroup of one amino acid and the amino group of another amino acid.Peptides also include peptide mimetics such as amino acids joined by anether as opposed to an amide bond.

The term “protease binding site” as used herein refers to an amino acidsequence that may be recognized and cleaved by a protease. The proteasebinding site contains a peptide bond that is hydrolyzed by the proteaseand the amino acid residues joined by this peptide bond are said to formthe cleavage site. The protease binding site and conformationdetermining regions form a contiguous amino acid sequence. The proteasebinding site may be an amino acid sequence that is recognized andcleaved by a particular protease. It is well known that variousproteases may cleave peptide bonds adjacent to particular amino acids.Thus, for example, trypsin cleaves peptide bonds following basic aminoacids such as arginine and lysine and chymotrypsin cleaves peptide bondsfollowing large hydrophobic amino acid residues such as tryptophan,phenylalanine, tyrosine and leucine. The serine protease elastasecleaves peptide bonds following small hydrophobic residues such asalanine. A particular protease, however, may not cleave every bond in aprotein that has the correct adjacent amino acid. Rather, the proteasesmay be specific to particular amino acid sequences which serve asprotease binding sites for each particular protease. Any amino acidsequence that comprises a protease binding site and may be recognizedand cleaved by a protease is a suitable protease receptor. Knownprotease binding sites and peptide inhibitors of proteases posses aminoacid sequences that are recognized by the specific protease they arecleaved by or that they inhibit. Thus known substrate and inhibitorsequences provide the basic sequences suitable for use as a proteasereceptor. A number of protease substrates and inhibitor sequencessuitable for use as protease binding sites are described in U.S. Pat.No. 6,037,137 which is incorporated herein by reference. One of skillwill appreciate that the protease substrates listed in U.S. Pat. No.6,037,137 is not a complete list and that other protease substrates orinhibitor sequences may be used.

Proteases (e.g., botulinum and tetanus toxins) cleave peptide bonds atspecific sequence sites on the proteins that “dock” neurotransmittersecretory vesicles to their cellular release sites (FIG. 45A, 45B). Whenone or more of these proteins is degraded in this fashion, secretion isblocked and paralysis results (FIG. 45C). It is known that relativelylow molecular weight peptides (˜15-35 amino acids) based on the normalprotein substrates of the botulinum toxins can be rapidly cleaved insolution by a toxin in a manner similar to the full-length protein. Suchexperiments have been described by Schmidt, J. J.; Stafford, R. G.;Bostian, K. A. “Type A botulinum neurotoxin proteolytic activity:development of competitive inhibitors and implications for substratespecificity at the S₁′ binding subsite” FEBS Lett., 1998, 435, 61-64 andShone, C. C.; Roberts, A. K. “Peptide substrate specificity andproperties of the zinc-endopeptidase activity of botulinum type Bneurotoxin” Eur. J. Biochem., 1994, 225, 263-270, both of which areincorporated herein by reference as if set forth herein. It has alsobeen demonstrated that these peptide substrates can retain high levelsof activity for both botulinum and tetanus toxins even when chemicallymodified by amino acid substitutions and fluorescence labeling (See alsoSoleihac, J.-M.; Comille, F.; Martin, L.; Lenoir, C.; Fournie-Zaluski,M.-C.; Roques, B. P. “A sensitive and rapid fluorescence-based assay fordetermination of tetanus toxin peptidase activity” Anal. Biochem., 1996,241, 120-127 and Adler, M.; Nicholson, J. D.; Hackley, B. E., Jr.“Efficacy of a novel metalloprotease inhibitor on botulinum neurotoxin Bactivity” FEBS Lett., 1998, 429, 234-238 both of which are incorporatedherein by reference).

For newly discovered proteases, or proteases of which the proteaserecognition sequence is not known, a suitable amino acid sequence foruse as the protease binding site may be determined experimentally. Thesynthesis of libraries of peptides and the use of these libraries todetermine a protease binding sequence for a particular protease isdescribed in U.S. Pat. No. 5,834,318 which is incorporated herein byreference. Generally, combinatorial libraries composed of between about2 to about 20 amino acids may be synthesized. These libraries may beused to screen for an interaction with the protease. Analysis of thesequences that bind to the protease may be used to determine potentialbinding sequences for use as a receptor for the protease.

The interaction of the receptor with a protease may be indicated by anindicator molecule coupled to the receptor or the polymeric resin. Inone embodiment, the indicator may be a chromophore or a fluorophore. Afluorophore is a molecule that absorbs light at a characteristicwavelength and then re-emits the light most typically at acharacteristic different wavelength. Fluorophores include, but are notlimited to rhodamine and rhodamine derivatives, fluorescein andfluorescein derivatives, coumarins and chelators with the lanthanide ionseries. A chromophore is a molecule which absorbs light at acharacteristic wavelength, but does not re-emit light.

In one embodiment, a peptide containing the cleavage sequence isimmobilized through a covalent or strong non-covalent bond to anaddressable site on a sensor array. In one embodiment, this may beaccomplished by coupling the peptide to a polymeric resin, as describedabove. The polymeric resin may be positioned in a cavity of a sensorarray, such as the sensor arrays described above. In some embodiments,different peptides containing different cleavage sequences for thevarious proteases may be immobilized at different array positions. Asample containing one or more proteases may be applied to the array, andpeptide cleavage may occur at specific array addresses, depending on thepresence of particular proteases. Alternatively, different peptidescontaining different cleavage sequences may be coupled to a singlepolymeric bead. In this manner, a single bead may be used to analyzemultiple proteases.

A variety of signaling mechanisms for the above described cleavagereactions may be used. In an embodiment, a fluorescent dye and afluorescence quencher may be coupled to the biopolymer on opposite sidesof the cleavage site. The fluorescent dye and the fluorescence quenchermay be positioned within the Forster energy transfer radius. The Forsterenergy transfer radius is defined as the maximum distance between twomolecules in which at least a portion of the fluorescence energy emittedfrom one of the molecules is quenched by the other molecule. Forsterenergy transfer has been described above. Before cleavage, little or nofluorescence may be generated by virtue of the molecular quencher. Aftercleavage, the dye and quencher are no longer maintained in proximity ofone another, and fluorescence may be detected (FIG. 62A). The use offluorescence quenching is described in U.S. Pat. No. 6,037,137 which isincorporated herein by reference. Further examples of this energytransfer are described in the following papers, all of which areincorporated herein by reference: James, T. D.; Samandumara, K. R. A.;Iguchi, R.; Shinkai, S. J. Am. Chem. Soc. 1995, 117, 8982. Murukami, H.;Nagasaki, T.; Hamachi, I.; Shinkai, S. Tetrahedron Lett., 34, 6273.Shinkai, S.; Tsukagohsi, K.; Ishikawa, Y.; Kunitake, T. J. Chem. Soc.Chem. Commun. 1991, 1039. Kondo, K.; Shiomi, Y.; Saisho, M.; Harada, T.;Shinkai, S. Tetrahedron. 1992, 48, 8239. Shiomi, Y.; Kondo, K.; Saisho,M.; Harada, T.; Tsukagoshi, K.; Shinkai, S. Supramol. Chem. 1993, 2, 11.Shiomi, Y.; Saisho, M.; Tsukagoshi, K.; Shinkai, S. J. Chem. Soc. PerkinTrans I 1993, 2111. Deng, G.; James, T. D.; Shinkai, S. J. Am. Chem.Soc. 1994, 116, 4567. James, T. D.; Harada, T.; Shinkai, S. J. Chem.Soc. Chem. Commun. 1993, 857. James, T. D.; Murata, K.; Harada, T.;Ueda, K.; Shinkai, S. Chem. Lett. 1994, 273. Ludwig, R.; Harada, T.;Ueda, K.; James, T. D.; Shinkai, S. J. Chem. Soc. Perkin Trans 2. 1994,4, 497. Sandanayake, K. R. A. S.; Shinkai, S. J. Chem. Soc., Chem.Commun. 1994, 1083. Nagasaki, T.; Shinmori, H.; Shinkai, S. TetrahedronLett. 1994, 2201. Murakami, H.; Nagasaki, T.; Hamachi, I.; Shinkai, S.J. Chem. Soc. Perkin Trans 2. 1994, 975. Nakashima, K.; Shinkai, S.Chem. Lett. 1994, 1267. Sandanayake, K. R. A. S.; Nakashima, K.;Shinkai, S. J. Chem. Soc. 1994, 1621. James, T. D.; Sandanayake, K. R.A. S.; Shinkai, S. J. Chem. Soc., Chem. Commun. 1994, 477. James, T. D.;Sandanayake, K. R. A. S.; Angew. Chem., Int. Ed. Eng. 1994, 33, 2207.James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S, Nature, 1995, 374,345.

The fluorophores may be linked to the peptide receptor by any of anumber of means well known to those of skill in the art. In anembodiment, the fluorophore may be linked directly from a reactive siteon the fluorophore to a reactive group on the peptide such as a terminalamino or carboxyl group, or to a reactive group on an amino acid sidechain such as a sulfur, an amino, a hydroxyl, or a carboxyl moiety. Manyfluorophores normally contain suitable reactive sites. Alternatively,the fluorophores may be derivatized to provide reactive sites forlinkage to another molecule. Fluorophores derivatized with functionalgroups for coupling to a second molecule are commercially available froma variety of manufacturers. The derivatization may be by a simplesubstitution of a group on the fluorophore itself, or may be byconjugation to a linker. Various linkers are well known to those ofskill in the art and are discussed below.

The fluorogenic protease indicators may be linked to a solid supportdirectly through the fluorophores or through the peptide backbonecomprising the indicator. In embodiments where the indicator is linkedto the solid support through the peptide backbone, the peptide backbonemay comprise an additional peptide spacer. The spacer may be present ateither the amino or carboxyl terminus of the peptide backbone and mayvary from about 1 to about 50 amino acids, preferably from 1 to about 20and more preferably from 1 to about 10 amino acids in length. The aminoacid composition of the peptide spacer is not critical as the spacerjust serves to separate the active components of the molecule from thesubstrate thereby preventing undesired interactions. However, the aminoacid composition of the spacer may be selected to provide amino acids(e.g. a cysteine or a lysine) having side chains to which a linker orthe solid support itself, is easily coupled. Alternatively the linker orthe solid support itself may be attached to the amino terminus of or thecarboxyl terminus.

In an embodiment, the peptide spacer may be joined to the solid supportby a linker. The term “linker”, as used herein, refers to a moleculethat may be used to link a peptide to another molecule, (e.g. a solidsupport, fluorophore, etc.). A linker is a hetero or homobifunctionalmolecule that provides a first reactive site capable of forming acovalent linkage with the peptide and a second reactive site capable offorming a covalent linkage with a reactive group on the solid support.Linkers as use din these embodiments are the same as the previouslydescribed linkers.

In an embodiment, a first fluorescent dye and a second fluorescent dyemay be coupled to the biopolymer on opposite sides of the cleavage site.Before cleavage, a FRET (fluorescence resonance energy transfer) signalmay be observed as a long wavelength emission. After cleavage, thechange in the relative positions of the two dyes may cause a loss of theFRET signal and an increase in fluorescence from the shorter-wavelengthdye (FIG. 62B). Examples of solution phase FRET have been described inFörster, Th. “Transfer Mechanisms of Electronic Excitation: Discuss.Faraday Soc., 1959, 27, 7; Khanna, P. L., Ullman, E. F.“4′,5′-Dimethoxyl-6-carboxyfluorescein: A novel dipole-dipole coupledfluorescence energy transfer acceptor useful for fluorescenceimmunoassays”, Anal. Biochem. 1980, 108, 156; and Morrison, L. E. “Timeresolved Detection of Energy Transfer: Theory and Application toImmunoassays”, Anal. Biochem. 1998, 174, 101, all of which areincorporated herein by reference.

In another embodiment, a single fluorescent dye may be coupled to thepeptide on the opposite side of the cleavage site to the polymericresin. Before cleavage, the dye is fluorescent, but is spatiallyconfined to the attachment site. After cleavage, the peptide fragmentcontaining the dye may diffuse from the attachment site (e.g., topositions elsewhere in the cavity) where it may be measured with aspatially sensitive detection approach, such as confocal microscopy(FIG. 62C). Alternatively, the solution in the cavities may be flushedfrom the system. A reduction in the fluorescence of the particle wouldindicate the presence of the analyte (e.g., a protease).

In another embodiment, a single indicator (e.g., a chromophore or afluorophore) may be coupled to the peptide receptor on the side of thecleavage site that remains on the polymeric resin or to the polymericresin at a location proximate to the receptor. Before cleavage theindicator may produce a signal that reflects the microevironmentdetermined by the interaction of the receptor with the indicator.Hydrogen bonding or ionic substituents on the indicator involved inanalyte binding have the capacity to change the electron density and/orrigidity of the indicator, thereby changing observable spectroscopicproperties such as fluorescence quantum yield, maximum excitationwavelength, or maximum emission wavelength for fluorophores orabsorption spectra for chromophores. When the peptide receptor iscleaved, the local pH and dielectric constants of the beads change, andthe indicator may respond in a predictable fashion. An advantage to thisapproach is that it does not require the dissociation of a preloadedfluorescent ligand (limited in response time by k_(off)). Furthermore,several different indicators may be used with the same receptor.Different beads may have the same receptors but different indicators,allowing for multiple testing for the presence of proteases.Alternatively, a single polymeric resin may include multiple dyes alongwith a single receptor. The interaction of each of these dyes with thereceptor may be monitored to determine the presence of the analyte.

Nucleases represent a number of families of enzymes that catalyticallyhydrolyze the phosphodiester bonds of nucleic acids. Nucleases may beclassified according to the nucleic acid that they are specific for.Ribonucleases (“RNases”) are specific for ribonucleic acids whiledeoxyribonucleases (“DNases”) are specific for deoxyribonucleic acids.Some enzymes will hydrolyze both ribonucleic acids and deoxyribonucleicacids. Nucleases may also be classified according to their point ofattack upon the nucleic acid. Nucleases that attack the polymer ateither the 3′ terminus or the 5′ terminus are known as exonucleases.Nucleases that attack the nucleic acid within the chain are calledendonucleases.

Restriction enzymes recognize short polynucleotide sequences and cleavedouble-stranded nucleic acids at specific sites within or adjacent tothese sequences. Approximately 3,000 restriction enzymes, recognizingover 230 different nucleic acid sequences, are known. They have beenfound mostly in bacteria, but have also been isolated from viruses,archaea and eukaryotes. Because many of these restriction enzymes areonly found in a particular organism, nucleic acids may be used as areceptor to determine if a particular organism is present in a sample byanalyzing for restriction enzymes. Restriction endonucleasesspecifically bind to nucleic acids only at a specific recognitionsequence that varies among restriction endonucleases. Since restrictionenzymes only cut nucleic acids in the vicinity of the recognitionsequence, a receptor may be designed that includes the recognitionsequence for the nuclease being investigated.

Most nucleases bind to and act on double stranded deoxyribonucleic acid(“DNA”). Restriction endonucleases are typically symmetrical dimers.Each monomeric unit binds to one strand of DNA and recognizes the firsthalf the DNA recognition sequence. Each monomer also typically cuts onestrand of DNA. Together, the dimer recognizes a palindromic DNA sequenceand cuts both strands of DNA symmetrically about the central point inthe palindromic sequence. Typically, each monomer of the restrictionendonucleases needs at least two specific nucleotides that itrecognizes, though in a few cases a restriction endonuclease monomeronly needs to bind to one specific nucleotide and two others with lessspecificity. This means that restriction endonucleases may recognize asequence of 4 nucleotides at a minimum, and generally recognizesequences that contain an even number of nucleotides (since the samesites are recognized by each monomer. Restriction endonucleases areknown that recognize 4, 6, or 8 nucleotides, with only a few 8-cuttersknown. Some restriction endonucleases bind to recognition sequences thathave an odd number of nucleotides (typically this is 5 or 7) with thecentral nucleotide specifically recognized or with some or strictspecificity for a central base pair. The origin and sequence specificityof hundreds of restriction endonucleases are known and can be found fromcatalogs available from New England Biolabs, Boston, Mass.; LifeTechnologies, Rockville, Md.; Promega Scientific, Madison, Wis., RoucheMolecular Biochemicals, Indianapolis, Ind.

In one embodiment, the presence of a nuclease may be detected by the useof a polynucleotide coupled to a polymeric resin. For the detection ofnucleases, the polynucleotide may be a double stranded deoxyribonucleicacid or a ribonucleic acid. Methods for synthesizing and/or attaching apolynucleotide to a polymeric resin are described, for example, in U.S.Pat. No. 5,843,655 which is incorporated herein by reference.“Polynucleotides” are herein defined as chains of nucleotides. Thenucleotides are linked to each other by phosphodiester bonds.“Deoxyribonucleic acid” is composed of deoxyribonucleotide residues,while “Ribonucleic acid” is composed of ribonucleotide residues.

The term “nuclease binding site” as used herein refers to apolynucleotide sequence that may be recognized and cleaved by anuclease. The nuclease binding site contains a phosphodiester bond thatis cleaved by the nuclease and the polynucleotide residues joined bythis phosphodiester bond are said to form the cleavage site.

For newly discovered nucleases, or nucleases of which the nucleaserecognition sequence is not known, a suitable polynucleotide sequencefor use as the nuclease binding site may be determined experimentally.Generally, combinatorial libraries of polynucleotides composed ofbetween about 2 to about 20 nucleotides may be synthesized. Thesynthesis of such libraries is described, for example, in U.S. Pat. No.5,843,655 which is incorporated herein by reference. These libraries maybe used to screen for an interaction with the nuclease. Analysis of thesequences that bind to the nuclease may be used to determine potentialbinding sequences for use as a receptor for the nuclease.

The interaction of the receptor with a nuclease may be indicated by anindicator molecule coupled to the receptor or the polymeric resin. Inone embodiment, the indicator may be a chromophore or a fluorophore.

In one embodiment, a polynucleotide containing the nuclease bindingsequence is immobilized through a covalent or strong non-covalent bondto an addressable site on a sensor array. In one embodiment, this may beaccomplished by coupling or synthesizing the polynucleotide on apolymeric resin, as described above. The polymeric resin may bepositioned in a cavity of a sensor array, such as the sensor arraysdescribed above. In some embodiments, different polynucleotidescontaining different cleavage sequences for the various nucleases may beimmobilized at different array positions. A sample containing one ormore nucleases may be applied to the array, and polynucleotide cleavagemay occur at specific array addresses, depending on the presence ofparticular nucleases. Alternatively, different polynucleotidescontaining different cleavage sequences may be coupled to a singlepolymeric bead. In this manner, a single bead may be used to analyzemultiple nucleases.

A variety of signaling mechanisms for the above described cleavagereactions may be used. In an embodiment, a fluorescent dye and afluorescence quencher may be coupled to the polynucleotide on oppositesides of the cleavage site. The fluorescent dye and the fluorescencequencher may be positioned within the Förster energy transfer radius.Before cleavage, little or no fluorescence may be generated by virtue ofthe molecular quencher. After cleavage, the dye and quencher are nolonger maintained in proximity of one another, and fluorescence may bedetected (FIG. 62A).

The fluorophores may be linked to the polynucleotide receptor by any ofa number of means well known to those of skill in the art. Examples ofmethods of attaching fluorophores and dyes to polynucleotides aredescribed in U.S. Pat. Nos. 4,855,225; 5,188,934, and 5,366,860 all ofwhich are incorporated herein by reference.

In another embodiment, a first fluorescent dye and a second fluorescentdye may be coupled to the polynucleotide receptor on opposite sides ofthe cleavage site. Before cleavage, a FRET (fluorescence resonanceenergy transfer) signal may be observed as a long wavelength emission.After cleavage, the change in the relative positions of the two dyes maycause a loss of the FRET signal and an increase in fluorescence from theshorter-wavelength dye (FIG. 62B).

In another embodiment, a single fluorescent dye may be coupled to thepolynucleotide receptor on the opposite side of the cleavage site to thepolymeric resin. Before cleavage, the dye is fluorescent, but isspatially confined to the attachment site. After cleavage, the nucleicacid fragment containing the dye may diffuse from the attachment site(e.g., to positions elsewhere in the cavity) where it may be measuredwith a spatially sensitive detection approach, such as confocalmicroscopy (FIG. 62C). Alternatively, the solution in the cavities maybe flushed from the system. A reduction in the fluorescence of theparticle would indicate the presence of the analyte (e.g., a nuclease).

In another embodiment, depicted in FIG. 62D, a single indicator (e.g., achromophore or a fluorophore) may be coupled to the polynucleotidereceptor on the side of the cleavage site that remains on the polymericresin or to the polymeric resin at a location proximate to thepolynucleotide receptor. Before cleavage the indicator may produce asignal that reflects the microevironment determined by the interactionof the receptor with the indicator. Hydrogen bonding or ionicsubstituents on the indicator involved in analyte binding have thecapacity to change the electron density and/or rigidity of theindicator, thereby changing observable spectroscopic properties such asfluorescence quantum yield, maximum excitation wavelength, or maximumemission wavelength for fluorophores or absorption spectra forchromophores. When the polynucleotide receptor is cleaved, the local pHand dielectric constants of the beads change, and the indicator mayrespond in a predictable fashion. An advantage to this approach is thatit does not require the dissociation of a preloaded fluorescent ligand(limited in response time by k_(off)). Furthermore, several differentindicators may be used with the same receptor. Different beads may havethe same receptors but different indicators, allowing for multipletesting for the presence of nucleases. Alternatively, a single polymericresin may include multiple dyes along with a single receptor. Theinteraction of each of these dyes with the receptor may be monitored todetermine the presence of the analyte.

In another embodiment, polynucleotide receptors may be used to determinethe presence of other types of analytes. It some instances,polynucleotide receptors will bind to small organic molecules. Thesesmall organic molecules may disrupt the action of nucleases upon thepolynucleotide receptor. Typically, the small molecules will occupy thepreferred binding site of the nuclease, inhibiting the action of thenuclease on the polynucleotide. Thus the presence of a small organicmolecule, which is known to bind to a specific polynucleotide, may bedetected by the observation of reduced nuclease activity at the specificpolynucleotide. An analogous methodology may be applied to apeptide-protease reaction.

In another embodiment, oligosaccharides may also be used to determinethe presence of analytes. In a system similar to those described abovefor peptides and polynucleotides, oligosaccharides may be coupled to apolymeric resin. In the presence of oligosaccharide cleaving agents(e.g., enzymes such as amylase, an enzyme that cleaves a long saccharidepolymer and disaccharide cleaving enzymes such as invertase,P-galactosidase, and lactase, to name a few) the oligosaccharide may becleaved. The cleavage of the oligosaccharide may be used to generate asignal. Methods for synthesizing and/or attaching oligosaccharides to apolymeric resin are described, for example, in U.S. Pat. Nos. 5,278,303and 5,616,698 which are incorporated herein by reference.

In another embodiment, an analyte may cause a change to a biopolymer,but not necessarily cleavage of the biopolymer, when the analyteinteracts with the biopolymer. The induced change may cause a detectablesignal to be generated. Typically, the binding or association ability ofan indicator molecule with a biopolymer is dependent upon the structureof the biopolymer. If the structure of the biopolymer is altered, theassociation of an indicator molecule may be significantly altered. Sucha change may be accompanied by a change in the signal produced by theindicator. For biopolymers many different types of enzymes may induce avariety of structural changes to the biopolymer which may alter thebinding site of an associated indicator molecule. Such changes may occurwithout cleavage of the biopolymer.

Alternatively, an indicator and a biopolymer may be coupled to apolymeric bead. The biopolymer may undergo a chemical reaction in thepresence of an analyte. This chemical reaction may also induce a changein the chemical structure of the indicator. The change in the chemicalstructure of the indicator may lead to a detectable change in theoptical properties of the particle, signaling the presence of theanalyte.

In one example, NAD and glucose may be coupled to a polymeric bead. Thissystem may be used to detect the presence of an carbohydrate modifyingenzyme. For example, the system may be used to detect the presence ofglucose dehydrogenase. In the presence of glucose dehydrogenase, glucosemay be consumed, and in the process would convert the coupled NAD intoNADH. NADH has both different UV absorbance and different fluorescenceproperties from NAD. These differences may be used to signal thepresence of glucose dehydrogenase in a fluid sample. Many other types ofenzymes may be detected in a similar manner. In an example, the proteasetrypsin was analyzed using an immobilized “sacrificial receptor” that iscleaved by trypsin, an event that results in modulation of afluorescence signal.

In an embodiment of a protease assay, a peptide that may be cleavedbetween two amino acids by the enzyme trypsin was immobilized. Thisimmobilization was accomplished by first conjugating many streptavidinmolecules to aldehyde-activated 6% agarose beads using a reductiveamination procedure. A biotin chemical group attached to theamino-terminus of the peptide was strongly bound by the immobilizedstreptavidin molecules, thereby immobilizing the peptide chains. Afluorescein group was attached to the carboxyl-terminus of the peptide,thereby making the bead highly fluorescent. Importantly, the immobilizedpeptide contains a cleavage site recognized by trypsin between thebiotin attachment site and the fluorescein, so that exposure of the beadto trypsin analyte causes release of fluorescent peptide fragments fromthe bead. This release may be visualized either as a decrease in thefluorescence at the bead, or by an increase in the fluorescence of thesurrounding solution (see FIG. 63).

Transmitting Chemical Information Over A Computer Network

Herein we describe a system and method for the collection andtransmission of chemical information over a computer network. Thesystem, in some embodiments, includes an analyte detection device(“ADD”) operable to detect one or more analytes or mixtures of analytesin a fluid containing one or more analytes, and computer hardware andsoftware operable to send and receive data over a computer network toand from a client computer system.

Chemical information refers to any data representing the detection of aspecific chemical or a combination of chemicals. These data may include,but are not limited to chemical identification, chemical proportions, orvarious other forms of information related to chemical detection. Theinformation may be in the form of raw data, including binary oralphanumeric, formatted data, or reports. In some embodiments, chemicalinformation relates to data collected from an analyte detection device.Such data includes data related to the color of the particles includedon the analyte detection device. The chemical information collected fromthe analyte detection device may include raw data (e.g., a color, RBGdata, intensity at a specific wavelength) etc. Alternatively the datamay be analyzed by the analyte detection device to determine theanalytes present. The chemical information may include the identities ofthe analytes detected in the fluid sample. The information may beencrypted for security purposes.

In one embodiment, the chemical information may be in LogicalObservation Identifiers Names and Codes (LOINC) format. The LOINC formatprovides a standard set of universal names and codes for identifyingindividual laboratory results (e.g. hemoglobin, serum sodiumconcentration), clinical observations (e.g. discharge diagnosis,diastolic blood pressure) and diagnostic study observations, (e.g.PR-interval, cardiac echo left ventricular diameter, chest x-rayimpression).

More specifically, chemical information may take the form of datacollected by the analyte detection system. As described above, ananalyte detection system may include a sensor array that includes aparticle or particles. These particles may be configured to produce adetectable signal in response to the presence or absence of an analyte.The signal may be detected using a detector. The detector may detect thesignal. The detector may also produce an output signal that containsinformation relating to the detected signal. The output signal may, insome embodiments be the chemical information.

In some embodiments, the detector may be a light detector and the signalproduced by the particles may be modulated light. The detector mayproduce an output signal that is representative of the detected lightmodulation. The output signal may be representative of the wavelength ofthe light signal detected. Alternatively, the output signal may berepresentative of the strength of the light signal detected. In otherembodiments, the output signal may include both wavelength and strengthof signal information.

In some embodiments, use of a light source may not be necessary. Theparticles may rely on the use of chemiluminescence, thermoluminescenceor piezoluminescence to provide a signal. In the presence of an analyteof interest, the particle may be activated such that the particlesproduce light. In the absence of an analyte, the particles may notexhibit produce minimal or no light. The chemical information may,therefore, be related to the detection or absence of a light produced bythe particles, rather than modulated by the particles.

The detector output signal information may be analyzed by analysissoftware. The analysis software may be configured to convert the rawoutput data to chemical information that is representative of theanalytes in the analyzed fluid system. The chemical information may beeither the raw data before analysis by the computer software or theinformation generated by processing of the raw data.

The term “computer system” as used herein generally describes thehardware and software components that in combination allow the executionof computer programs. The computer programs may be implemented insoftware, hardware, or a combination of software and hardware. Computersystem hardware generally includes a processor, memory media, andinput/output (I/O) devices. As used herein, the term “processor”generally describes the logic circuitry that responds to and processesthe basic instructions that operate a computer system. The term “memorymedium” includes an installation medium, e.g., a CD-ROM, floppy disks; avolatile computer system memory such as DRAM, SRAM, EDO RAM, Rambus RAM,etc.; or a non-volatile memory such as optical storage or a magneticmedium, e.g., a hard drive. The term “memory” is used synonymously with“memory medium” herein. The memory medium may comprise other types ofmemory or combinations thereof. In addition, the memory medium may belocated in a first computer in which the programs are executed, or maybe located in a second computer that connects to the first computer overa network. In the latter instance, the second computer provides theprogram instructions to the first computer for execution. In addition,the computer system may take various forms, including a personalcomputer system, mainframe computer system, workstation, networkappliance, Internet appliance, personal digital assistant (PDA),television system or other device. In general, the term “computersystem” can be broadly defined to encompass any device having aprocessor that executes instructions from a memory medium.

The memory medium preferably stores a software program or programs forthe reception, storage, analysis, and transmittal of informationproduced by an Analyte Detection Device (ADD). The software program(s)may be implemented in any of various ways, including procedure-basedtechniques, component-based techniques, and/or object-orientedtechniques, among others. For example, the software program may beimplemented using ActiveX controls, C++ objects, JavaBeans, MicrosoftFoundation Classes (MFC), or other technologies or methodologies, asdesired. A central processing unit (CPU), such as the host CPU, forexecuting code and data from the memory medium includes a means forcreating and executing the software program or programs according to themethods, flowcharts, and/or block diagrams described below.

A computer system's software generally includes at least one operatingsystem such as Windows NT, Windows 95, Windows 98, or Windows ME (allavailable from Microsoft Corporation); or Mac OS and Mac OS X Server(Apple Computer, Inc.), MacNFS (Thursby Software), PC MACLAN (MiramarSystems), or real time operating systems such as VXWorks (Wind RiverSystems, Inc.), QNX (QNX Software Systems, Ltd.), etc. The foregoing areall examples of specialized software programs that manage and provideservices to other software programs on the computer system. Software mayalso include one or more programs to perform various tasks on thecomputer system and various forms of data to be used by the operatingsystem or other programs on the computer system. Software may also beoperable to perform the functions of an operating system (OS). The datamay include but is not limited to databases, text files, and graphicsfiles. A computer system's software generally is stored in non-volatilememory or on an installation medium. A program may be copied into avolatile memory when running on the computer system. Data may be readinto volatile memory as the data is required by a program.

A server program may be defined as a computer program that, whenexecuted, provides services to other computer programs executing in thesame or other computer systems. The computer system on which a serverprogram is executing may be referred to as a server, though it maycontain a number of server and client programs. In the client/servermodel, a server program awaits and fulfills requests from clientprograms in the same or other computer systems.

Examples of computer programs that may serve as servers include: WindowsNT (Nicrosoft Corporation), Mac OS X Server (Apple Computer, Inc.),MacNFS (Thursby Software), PC MACLAN (Miramar Systems), etc

A web server is a computer system which maintains a web site browsableby any of various web browser software programs. As used herein, theterm ‘web browser’ refers to any software program operable to access websites over a computer network.

An intranet is a network of networks that is contained within anenterprise. An intranet may include many interlinked local area networks(LANs) and may use data connections to connect LANs in a wide areanetwork (WAN). An intranet may also include connections to the Internet.An intranet may use TCP/IP, HTTP, and other Internet protocols.

An extranet, or virtual private network, is a private network that usesInternet protocols and public telecommunication systems to securelyshare part of a business' information or operations with suppliers,vendors, partners, customers, or other businesses. An extranet may beviewed as part of a company's intranet that is extended to users outsidethe company. An extranet may require security and privacy. Companies mayuse an extranet to exchange large volumes of data, share productcatalogs exclusively with customers, collaborate with other companies onjoint development efforts, provide or access services provided by onecompany to a group of other companies, and to share news of commoninterest exclusively with partner companies.

Connection mechanisms included in a network may include copper lines,optical fiber, radio transmission, satellite relays, or any other deviceor mechanism operable to allow computer systems to communicate.

As used herein, ADD refers to any device or instrument operable todetect one or more specific analytes or mixtures of analytes in a fluidsample, wherein the fluid sample may be liquid, gaseous, solid, asuspension of a solid in a gas, or a suspension of a liquid in a gas.More particularly, an ADD includes a sensor array, light and detector asis described herein.

As illustrated in FIG. 64, an ADD 102 is operable to analyze a fluidsample and detect one or more analytes in the sample, producing outputdata specifying the results of the detection process. ADD 102 may beoperable to connect to a computer network 104, such as the Internet. Asused herein, “computer network” may refer to any type of intranet orextranet network which connects computers and/or networks of computerstogether, thereby providing connectivity between various systems forcommunication there between, using various network communicationprotocols, such as TCP/IP, FTP, HTTP, HTTPS, etc. ADD 102 may executesoftware to communicate with other computer systems connected to network104.

A client computer 106 may also be connected to network 104. The clientsystem 106 may be a computer system, network appliance, Internetappliance, personal digital assistant (PDA) or other system. Clientcomputer system 106 may execute software to communicate with ADD 102,thus facilitating transmission of chemical data from the ADD 102 toclient computer system 106 and vice versa.

In one embodiment, the ADD may execute software operable to transmitchemical data via any of various communication protocols over thenetwork to one or more recipient client computer systems and to receiveresponses from the recipient client computers. These protocols mayinclude, but are not limited to, TCP/IP, FIP, HTTP, and ITTPS. As statedabove, the chemical information may be encrypted for security purposes.

As FIG. 65 illustrates, in step 110 an ADD 102 may be used to analyze achemical sample and detect one or more particular analytes orcombinations of analytes, producing output data comprising the resultsof the detection process. As stated above, this information may be in avariety of forms and formats, including binary, alphanumeric, reports,etc. In one embodiment, the ADD is configured to detect optical signalsproduced by the reaction of the analyte with a sensor array ofparticles. The optical signals may be converted to output datarepresentative of the optical signal.

In step 112 the chemical information may be transmitted over network 104to one or more client computer systems 106 using any of a variety ofnetwork communication protocols as described herein.

In step 114 one or more client computer systems 106 may each optionallytransmit a response back to ADD 102. The response may include, but isnot limited to, a request for additional information, a confirmation ofreceived data, or a transmittal of chemical information back to the ADD.

Some embodiments of the ADD include a light source, a sensor array, anda detector. The sensor array, in some embodiments, is formed of asupporting member which is configured to hold a variety of chemicallysensitive particles (herein referred to as “particles”) in an orderedarray. The particles are, in some embodiments, elements which willcreate a detectable signal in the presence of an analyte. The particlesmay produce optical (e.g., absorbance or reflectance) orfluorescence/phosphorescent signals upon exposure to an analyte.Examples of particles include, but are not limited to, functionalizedpolymeric beads. The particles may include a receptor molecule coupledto a polymeric bead. The receptors, in some embodiments, are chosen forinteracting with analytes. The interaction may take the form of abinding/association of the receptors with the analytes. The supportingmember may be made of any material capable of supporting the particles,while allowing the passage of the appropriate wavelengths of light. Thesupporting member may include a plurality of cavities. The cavities maybe formed such that at least one particle is substantially containedwithin the cavity. Upon contact of the beads with a fluid sample, adetectable optical signal may be generated by the receptor molecules'reactions with the one or more analytes in the sample.

In an alternate form of the invention, ADD 102 may be operable to uploadchemical data directly to a local computer system 108, for example, by acommunications link such as a serial data connection, wireless datalink, modem, floppy drive, etc., as depicted in FIG. 66. Local computersystem 108 may be connected to the computer network 104, as may beclient computer system 106. The local computer system 108 may havesoftware executable to transmit chemical information to the clientcomputer system 106 and to receive response information back from theclient computer system 106, and client computer system 106 may havesoftware executable to receive chemical information and to transmit aresponse back to local computer system 108 or to one or more receivingcomputer systems 107.

As FIG. 67 illustrates, in step 210 an ADD 102 may be used to analyze achemical sample and detect one or more particular analytes orcombinations of analytes, producing output data comprising the resultsof the detection process.

In step 212 chemical information may be uploaded to a local computersystem 108, such as by a communication link as described above. Localcomputer system 108 is connected to the network 104 and may use asoftware program executable to transmit the chemical information overnetwork 104.

As shown in step 214, the chemical information may be transmitted overnetwork 104 to one or more client computer systems 106 using any of avariety of network communication protocols, such protocols beingfamiliar to one skilled in the network communication art.

In step 216 client computer system 106 may optionally transmit aresponse back to local computer system 106 over network 104, or to oneor more receiving computer systems 107.

As shown in FIG. 68, ADD 102 may connect to a server 302, eitherdirectly, as with a communication link, or remotely, via computernetwork 104. The server 302 is operable to receive and store thechemical information, and to make the chemical information available toclient computer systems 106 also connected to network 104. The server302 may be any of a variety of servers. For example, server 302 may be aweb server, wherein the server is operable to maintain a web site,accessible by client computer systems 106 with browser software. Theuser of client computer system 106 may view and/or download the chemicalinformation from server 302 using the browser software. As anotherexample, the server may be an FTP server, in which case the user ofclient computer system 106 may be able to transfer the chemicalinformation from server 302 to client computer system 106 using an FTPsoftware program. As yet another example, server 302 may allow remotelogin to an account by client computer system 106, wherein the accounthas been established for use by the user of client computer system. Theuser of client computer system 106 may then view, edit, or transfer thechemical information as needed. Client computer system 106 may thenoptionally transmit a response back to server 302, which may then beaccessed by the ADD. Client computer system 106 may also transmit theresponse information to one or more additional client computer systems107. In all of these embodiments, security measures may be employed toprotect the identity of the users, as well as the privacy and integrityof the information. Such security measures may include secure login,encryption, private communication lines, and other security measures.

As FIG. 69 illustrates, in step 310 an ADD 102 may be used to analyze achemical sample and detect one or more particular analytes orcombinations of analytes, producing output data comprising the resultsof the detection process.

In step 312 the chemical information may be uploaded to a server 302,either directly, as by communication link, or via the computer network104. There, the chemical information may be stored.

As described above, server 302 is connected to network 104, as is theclient computer system 106. In step 314 client computer system 106 mayconnect to server 302 over network 104.

As shown in step 316, chemical information may be transmitted by server302 over the network to client computer system 106 using any of avariety of network communication protocols, such as TCP/IP, FTP, HTTP,HTTPS, etc.

In step 318 client computer system 106 may optionally transmit responseinformation back to server 302, which then may be accessed by ADD 102 toretrieve the response information, or to one or more additional clientcomputer systems 107.

In one embodiment, server 302 is a web server operable to maintain a website. When a client computer system accesses the web site of web server120, web server 120 provides various data and information to the clientbrowser on client system 106, possibly including a graphical userinterface (GUI) that displays the information, descriptions of theinformation, and/or other information that might be useful to the usersof the system.

In some embodiments, the ADD may include an electronic controller, asdescribed herein. The electronic controller may allow the ADD to beoperated by a client computer that is coupled to the electroniccontroller. The client computer may include software that provides theuser information regarding the operation of the ADD. The client computermay allow the user of the client computer to issue commands that allowoperation of the ADD from the electronic controller. The issued commandsmay be converted to control signals. The control signals may be receivedby the electronic controller. The electronic controller may operatecomponents of the ADD in response to the received control signals.

The client computer may be coupled directly to the ADD. Alternatively,the client computer may be coupled to the ADD via a computer network. Inthis embodiment, an operator may be in a different location than thelocation of the ADD. By sending control signals over the computernetwork, the operator may remotely control the operation of the ADD. TheADD may also be configured to transmit the obtained chemical informationback to the client computer via the computer network.

In another embodiment, the client computer may be coupled to the ADD viaa server, as described before. The client computer may be configured toreceive and/or transmit information to the ADD. In one embodiment, theADD may be configured to receive control signals from the clientcomputer via the server. The operation of the ADD may, therefore, becontrolled via a client computer through a server. As discussed beforethe ADD may also transmit chemical information back to the clientcomputer via the server.

In one embodiment, the ADD may be used to detect and identify one ormore analytes in the blood serum of an animal or person in a remotelocation. The ADD may be configured with the appropriate detectioncomponents and software to detect the presence of any of a great numberof different analytes. The serum sample may be processed by the ADD andthe results, the chemical information, transmitted to a client computersystem residing at a diagnosis center (e.g. a veterinary hospital ormedical office). There a medical expert may receive the chemicalinformation and interpret it to diagnose the probable cause and/orsource of the detected analytes. The medical expert may use thisinformation to make a diagnosis of the patients medical condition. Basedon the diagnosed medical condition, the medical expert may alsoprescribe medication for the treatment of the medical condition. Thisinformation may be transmitted back to the ADD over a computer networkor a server. The information may also be transmitted to other clientcomputer systems that are linked using a computer network or a server.For example, the medical expert may transmit a prescription to the ADDand to a client computer system at a pharmacy, which may then fill theprescription.

In another example of the use of an embodiment of the invention, ADD 102is used to detect pollutants in a water supply, e.g. a remote lake orstream. ADD 102 processes the water sample, and the resulting detectioninformation is transmitted to the web server 302 at an EnvironmentalMonitoring station. The pollution information may include bothidentification and concentrations of the chemicals detected. There, theinformation may be input into a software program which updates a map ofarea waterways with pollution information superimposed thereon. Theupdated map may be displayed on a web site for use by interestedparties.

In another embodiment, the invention may be used for home drugmetabolite tracking. Detection and measurement of blood and urinecomponents by ADD 102 may be used to track the appearance anddisappearance of various drug components after dosing. The user of ADD102 may upload the test results to a client computer system 106 used bya health care professional. The upload may be accomplished either bytransferring the information to a local computer system, thentransmitting over network 104 to client computer system 106 of thehealth professional, or directly from ADD 102 to client system 106 overnetwork 104 (e.g., through an internal modem similar to those used inPDAs or other hand-held computing devices). Results of the tests may beexamined either by human or software, and recommendations made to eithercontinue current drug protocol or to modify dosing to achieve a desiredmetabolite profile. These recommendations may then be transmitted backto the user of ADD 102, either via the local computer system, ordirectly, depending upon the ADD communication capabilities. Throughthis method, accurate determinations of doses needed to achieveeffective treatment while avoiding dangerous over-medication may bepossible. This offers a revolutionary change from current approaches, inwhich most or all people in a population are treated identically,regardless of ethnicity, gender, age, and medication with other drugs.Some studies indicate that effective and toxic dose levels can varysignificantly for these different subgroups of patients. By providing asimple and fast means for frequent metabolite analysis and evaluation,network uploading of ADD detection results, and possible subsequentdownloading of recommendations can open fundamentally new ways to treatpatients.

According to another embodiment, the method and system may be used forhome blood component analysis by a patient. Similar to drug metabolitetracking, the results of analyses for natural blood components (e.g.,glucose, insulin, cholesterol (LDLs/HDLs), triglycerides,prostate-specific antigen, and other indicators of health state) may beuploaded to a client computer system 106 of a health care professional.Examination of test results could then be used for diagnosis, or atleast early-warning screening for possible pathologies. Recommendationsfor action (e.g., drug use or scheduling of appointment) may then betransmitted back to the patient's ADD 102 or local computer system 106or phoned to the patient. Again, the potential for simple, fast, andfrequent measurements may provide safeguards for patients in certainrisk groups (e.g., diabetes), who would otherwise need to make frequenttrips to the lab or at the minimum would otherwise have to manually callin/email home test results—a far less reliable approach than automateduploading of the chemical information by the ADD following its analysis.

Another embodiment of the invention pertains to field-testing ofenvironmental conditions. Automated sensing of environmental conditions,including the presence of natural chemicals, industrial wastes, andbiological/chemical warfare agents is possible using an embodiment ofthe invention. Uploading of test results via radio transmission mayprovide remote sensing capabilities, and may provide responsecapabilities through human or central computer directed action. Responseinstructions may then be downloaded either to the sensing site or toanother strategic response position. Such a system may be useful, forexample, in determining the presence of toxins in a public water supply,and the subsequent centralized-directed cessation of water flow from thesupply pool.

In one embodiment, data may be collected from a remote location and thedata transmitted to a third party at an alternate location. A sample maybe provided to a replaceable sensor cartridge having multiple analytesensors and being configured as part of the testing device. The samplemay be from a subject (e.g. a patient) and provided to the replaceablesensor cartridge by an operator. Data regarding the sample, from themultiple analyte sensor, may be transmitted to a central data service.At the central data service, one or more tests may be performed on theelectronic data using the central data service. After the tests havebeen performed, an electronic message may be transmitted to a thirdparty, remote from the central data service. The information may includethe results of one or more of the tests. In some embodiments, the testsmay be selected by the third party. After the third party has receivedthe results of the test the appropriate response (e.g. treatment in thecase of medical diagnostics) may be selected.

A sensor array system may also be used for remote diagnostic screening.In one embodiment, a medical practitioner may prescribe a treatment to apatient during a visit. The medical practitioner may also wish tomonitor the quantity of treatment for the patient. In one embodiment,the patient may provide a sample to a remote analyte testing device. Theresults produced by the analyte testing device may be transmitted to acentral data service center. The central data service center may performan analysis of the data and make recommendations to the patient tomodify, maintain or cease the current treatment. The treatment may be inthe form of medication, or an applied medical procedure. For medicationtreatments, the central data center may also note if any othermedications are present. If so, the central data service center mayadvise the patient and/or practitioner of possible adverse druginteractions. Allergic reactions may also be detected and reported inthis manner.

Office visits may also be scheduled using the sensor array system. Datacollected from a patients sample may be sent from the sensor arraysystem to a central data service. The electronic test data may beanalyzed at the central data service. The results of the tests may betransmitted to a medical practitioner. The medical practitioner, uponreview of the test results may schedule an appointment for the patient.The subject may be notified of the need for an office visit through thecentral data service. Alternatively, the medical practitioner may decidethat an office visit is unnecessary, but wish to alter the treatment.The medical practitioner may, either directly or indirectly (through thecentral data service) inform the patient of the change of treatment.

Diagnostic Uses of the Sensor Array System

One of the largest markets for the health care industry is thediagnostic market. The worldwide market for diagnostic products is inthe range of about $20 billion a year. Much of this market is driven bythe current managed health care environment. The importance ofdiagnostics in the reduction of health care costs have created a needfor early and less expensive diagnosis. Generally, an early and accuratediagnosis may lead to early prognosis, reduced unnecessary testing andsignificantly lower health costs. This is especially true for animalmanagement. Animal diagnosis tends to be less accurate because of thetypes of testing being used. Instead of performing detailed diagnostictesting of animals, many animal care workers tend to prepare preventivemixtures which include a number of drugs for a variety of potentialdiseases that the animals may or may not have. These mixtures areexpensive and may, in the case of antibiotics, promote antibioticresistant pathogens.

The previously described sensor array system may be used for a widevariety of diagnostic testing for both animals and humans. As describedbefore, the sensor array may include a variety of particles that arechemically sensitive to a variety of types of analytes. In oneembodiment, the particles may be composed of polymeric beads. Attachedto the polymeric beads may be at least one receptor. The receptors maybe chosen based on their binding ability with the analyte of interest.

The sensor array may be adapted for use with a variety of bodily fluids.Blood and urine are the most commonly used bodily fluids for diagnostictesting. Other body fluids such as saliva, sweat, mucus, semen, and milkmay also be analyzed using a sensor array. The analysis of most bodilyfluids will, typically, require a filtration of the material prior toanalysis. For example, cellular material and proteins may need to beremoved from the bodily fluids. As previously described, theincorporation of filters onto the sensor array platform, may allow theuse of a sensor array with blood samples. These filters may also work ina similar manner with other bodily fluids, especially urine.Alternatively, a filter may be attached to a sample input port of thesensor array system, allowing the filtration to take place as the sampleis introduced into the sensor array.

In one embodiment, a sensor array may be customized for use as animmunoassay diagnostic tool. Immunoassays rely on the use of antibodiesor antigens for the detection of a component of interest. In nature,antibodies are produced by immune cells in response to a foreignsubstance (generally known as the “antigen”). The antibodies produced bythe immune cell in response to the antigen will typically bind only tothe antigen that elicited the response. These antibodies may becollected and used as receptors that are specific for the antigen thatwas introduced into the organism.

In many common diagnostic tests, antibodies are used to generate anantigen specific response. Generally, the antibodies are produced byinjecting an antigen into an animal (e.g., a mouse, chicken, rabbit, orgoat) and allowing the animal to have an immune response to the antigen.Once an animal has begun producing antibodies to the antigen, theantibodies may be removed from the animal's bodily fluids, typically ananimal's blood (the serum or plasma) or from the animal's milk.Techniques for producing an immune response to antigens in animals arewell known.

Once removed from the animal, the antibody may be coupled to a polymericbead. The antibody may then acts as a receptor for the antigen that wasintroduced into the animal. In this way, a variety of chemicallyspecific receptors may be produced and used for the formation of achemically sensitive particle. Once coupled to a particle a number ofwell known techniques may be used for the determination of the presenceof the antigen in a fluid sample. These techniques includeradioimmunoassay (RIA) and enzyme immunoassays such as enzyme-linkedimmunosorbent assay (ELISA). ELISA testing protocols are particularlysuited for the use of a solid support such as polymeric beads. The ELISAtest typically involves the adsorption of an antibody onto a solidsupport. The antigen is introduced and allowed to interact with theantibody. After the interaction is completed a chromogenic signalgenerating process is performed which creates an optically detectablesignal if the antigen is present. Alternatively, the antigen may bebound to the solid support and a signal generated if the antibody ispresent. Immunoassay techniques have been previously described, and arealso described in the following U.S. Pat. Nos. 3,843,696, 3,876,504,3,709,868, 3,856,469, and 4,567,149 all of which are incorporated byreference.

In one embodiment, an immunoassay sensor array may be used for thediagnosis of bacterial infections in animals. Many animals suffer from avariety of bacterial, viral, parasitic, and/or fungal diseases that maybe unmonitored or not specifically diagnosed by the animals caretakers.Bacterial infections may be particularly troublesome since bacterialinfections tend to cause a number of different health problems, some ofwhich effect the quality of products produced from the animals. It isdesirable for the animal caretakers to diagnosis and treat such problemsas quickly as possible. The testing of animals, especially animals suchas chickens and cattle, may be difficult due to the large number ofindividual animals in the flock or herd. A diagnostic tool for animaltesting should be easy to use, accurate, quick and inexpensive. Such atool would allow better animal health management, especially for largecollections of animals.

For example, mastitis is a common bacterial infection that occurs in theudders of cows. The presence of the mastitis causing bacteria in cowsmay render the milk produced by the cows unsuitable for sale. Oncedetected, the treatment will involve the use of a mixture antibioticsthat also renders the milk unusable for a period of days. This canresult in a tremendous financial loss for the owners of the cows,especially if the infection spreads to the other cows of the herd.

Current mastitis detection includes daily observation of the bulk tanksomatic cell count. The bulk tank represent the bulk milk collected frommany different cows from the herd. The somatic cell count is a measureof any inflammatory blood cells present in the milk of the cow, thus itis a measure of any inflammatory process that may have affected theudder of the cow. The somatic cell count offers a method of screeningfor potential problems in both the herd and the individual cows, but aconfirmation test is necessary for a definitive diagnosis of mastitis.The confirmation tests typically involve culturing the milk andanalyzing the milk for the particular strains of bacteria that causemastitis. This process can take from 1 to 2 days to complete. Meanwhile,the communal use of milking machines may cause the infection to spreadwithin the herd.

The sensor array system described herein may be used to improve thediagnostic procedures for testing milk samples for cows. In oneembodiment, antibodies that are specific for the bacteria that causemastitis may be bound to the receptors. Immunoassays for the detectionof mastitis are described in U.S. Pat. No. 5,168,044 which isincorporated by reference. Using the testing protocols previouslydescribed, the sensor array system may be used to detect the presence ofmastitis causing bacteria in any of the bodily fluids of a cow. Theimmunoassay is typically faster, (i.e., completed in hours instead ofdays) and may allow rapid sampling of individual members of the herd. Ingeneral, the immunoassays are much more accurate than cell culturemethods, which tend to give false positive results.

Another advantage of using a sensor array system, is that multiplebacterial strains may be analyzed simultaneously. Cow milk, as well asother bodily fluids, may include other bacteria that may potentiallycause health problems for the animal. For example, a variety ofgram-positive bacteria such as staphylococcus and streptococcus andgram-negative bacteria such as coliforms (e.g., E. Coli), Proteus andPseudomonas may also be present in the fluid sample. Typically, mastitistests ignore these bacteria, or in some cases, may confuse the presenceof these bacteria for the mastitis causing bacteria. In one embodiment,a sensor array may include multiple particles, each particle including areceptor that is specific for a particular bacterial strain. In a singletest, all of the bacteria present in the animal may be detected. This isparticularly important for determining the proper treatment of theanimal. By identifying the strains of bacteria present in the animal'ssample the appropriate antibiotics may be chosen for a treatment. Thismay help to avoid the proliferation of antibiotic resistant pathogensdue to unnecessary use of antibiotics.

While described in detail for the detection of mastitis in cows, itshould be understood that the above described method of detectingbacteria in bodily fluids may be applied to a variety of differentbacteria found in both animals and/or humans. The sensor array wouldonly need to be modified with respect to the type of antibodies (orantigen) that are used for the testing procedure.

Additionally, bacteria in soil and grain samples may also be detectedusing an immunoassay procedure. In the case of soils and grains, anextraction of these mediums with a suitable solvent may be requiredprior to analysis. For example, a grain sample may be soaked in waterand the undissolved material filtered out before the water is analyzed.The analysis of the water may then take place using any of theprocedures for fluid samples previously described and the presence ofbacteria in the grain (or soil) may be determined.

In one embodiment, the sensor array is used to detect Mycobacteriumtuberculosis, the causative agent of tuberculosis. Immunoassays for thedetection of Mycobacterium tuberculosis are described in U.S. Pat. No.5,631,130 which is incorporated by reference.

Many animals also suffer from a variety of parasitic diseases. Parasiticdiseases may, occasionally, appear in humans as well. For example, oneof the most prevalent parasitic disease found in dogs is heartworms.Heartworms are caused by the D. immitis parasite. The early detection ofthe presence of this parasite in a dog is important. If caught at anearly stage, the parasite may be treated with the use of drugs beforeany permanent damage to the heart is caused. A number of tests may beused for the detection of a heartworm infection. Those that are mostapplicable for the sensor array system are based on immunoassays. Onetest, known as the indirect fluorescent antibody test is specific forantibodies produced by the dog against the heartworm microfilaria.Another test utilizes an ELISA based screening for detecting circulatingworm antigen. Both of these immunoassay tests have a high degree ofspecificity for the detection of heartworms.

The previously described sensor array may be adapted for the detectionof heartworms using either of these well know techniques. Additionally,other parasitic infections may be simultaneously analyzed for by the useof the additional particles which include receptors for other types ofparasitic infections, including protozoan infections. Alternatively, amixture of particles that are specific for either parasites or bacteriamay be incorporated into a single sensor array unit. Since the analytesfor bacteria and parasites tend to be found in the same bodily fluids(e.g., blood), the use of such a sensor array would allow the diagnosisof potential bacterial and parasitic diseases for an animal (or a human)to be simultaneously detected. While the above test has been describedwith respect to a dog, it should be understood that the testingprocedure would be applicable to other animals and humans.

Another source of disease in humans and animals is from viralinfections. For example, feline leukemia is a viral infection that,until recently, was the most common fatal disease of cats. The diseaseis primarily caused by the exposure of the cat to the feline leukemiavirus (FeLV). The feline leukemia virus may be detected usingimmunoassay techniques. Three major tests have been used to determinethe presence of FeLV. The blood ELISA test is the most accurate, andwill detect the presence of FeLV at any stage of infection. FeLV antigenmay be used as a receptor that binds FeLV. An older test is based on aindirect fluorescent antibody (IFA) test for antibodies that areproduced against FeLV. A third test is a tears/saliva ELISA test. TheIFA and tear/saliva ELISA tests are only accurate in the late stages ofthe disease. As described above, the attachment of the appropriateantibodies or antigens on the particle will allow any of these testingprocedures to be performed using the sensor array system.

The HIV virus and the hepatitis C virus (togovirus and calicivirus) areexamples of viruses that humans may be tested for. These viruses aremost commonly detected using an ELISA testing method. The ELISA testingmethods for HIV or hepatitis C look for antibodies in the bodily fluidsof the person being tested. The most commonly analyzed bodily fluidsused for these tests are blood and saliva. The attachment of theappropriate antigens on a particle will allow any of these testingprocedures to be performed using the sensor array system. An advantageof the use of a sensor array for the detection of viruses in humans, isthat many other pathogens may be simultaneously analyzed for. Forexample, viral infections from other viruses (e.g., hepatitis A,hepatitis B, human herpesvirus-8, cytomegalovirus, varicella zostervirus, etc.) and other pathogens (e.g., Pneumocystis carinii, Toxoplasmagondii, Mycobacterium avium, Mycobacterium intracellulare, Treponemapallidum, etc.) may be detected simultaneously with HIV and/or hepatitisC by the use of multiple particles with the appropriate antibodies orantigens. Pneumocystis carinii, Toxoplasma gondii, Mycobacterium avium,Mycobacterium intracellulare, cytomegalovirus, human herpesvirus-8, andvaricella zoster virus are organisms that cause infections inimmunocompromised patients. Treponema pallidum is the bacteria thatcauses syphilis. Immunoassays for the detection of Pneumocystis cariniiare described in U.S. Pat. No. 4,925,800 which is incorporated byreference. Immunoassays for the detection of Toxoplasma gondii,cytomegalovirus, Herpes simplex virus, and Treponema pallidum aredescribed in U.S. Pat. No. 4,294,817 which is incorporated by reference.Immunoassays for the detection of Toxoplasma gondii are also describedin U.S. Pat. No. 5,965,590 which is incorporated by reference.Immunoassays for the detection of Hepatitis B use Hepatitis B surfaceantigen as a receptor.

It should be understood that parasitic, viral and bacterial infectionsmay all be analyzed at substantially the same time using the sensorarray system. The sensor array system may include all of the necessaryreagents and indicators required for the visualization of each of thesetests. In addition, the sensor array may be formed such that thesereagents are compartmentalized. In this manner, the reagents requiredfor a viral tests may be isolated from those used for a bacterial test.The sensor array may offer a complete pathogen analysis of an animal orpersons bodily fluid with a single test.

The presence of fungus in grains may also be detected using a sensorarray system. The fungus in grains may be removed using an extractiontechnique. The samples may be analyzed with a sensor array system whichincludes particles that are sensitive to the presence of a variety offungi. In this was, the fungi present in a grain sample may bemonitored.

Diagnostic tests have also been used for the detection of variousorganic molecules in humans and animals. These molecules may be detectedby a variety of testing procedures, including, but not limited to,immunoassay techniques, enzyme binding techniques, and syntheticreceptors.

The concentration of glucose in human blood is commonly measured forpeople with diabetes. The measurement of the blood glucose level may beperformed more than 5 times a day for some individuals. Currentlyavailable home testing relies, primarily, on a blood test for thedetermination of the concentration of glucose. The determination ofglucose is typically determined by the enzymatic decomposition ofglucose. Some methods for the determination of glucose in blood aredescribed in U.S. Pat. Nos. 3,964,974 and 5,563,042 which areincorporated by reference.

Cholesterol is also a common constituent of blood that is frequentlymonitored by people. As with glucose, a number of home testing kits havebeen developed that rely on the use of an enzyme based testing methodfor the determination of the amount of cholesterol in blood. A methodfor the determination of cholesterol in blood is described in U.S. Pat.No. 4,378,429 which is incorporated by reference.

The triglyceride level in blood is also commonly tested for because itis an indicator of obesity, diabetes, and heart disease. A system forassaying for triglycerides in bodily fluids is described in U.S. Pat.No. 4,245,041 which is incorporated by reference.

The concentration of homocysteine may be an important indicator ofcardiovascular disease and various other diseases and disorders. Varioustests have been constructed to measure the concentration of homocysteinein bodily fluids. A method for the determination of homocysteine inblood, plasma, and urine is described in U.S. Pat. No. 6,063,581 whichis incorporated by reference.

Cholesterol, triglyceride, homocysteine, and glucose testing may beperformed simultaneously using the sensor array system. Particles thatare sensitive to either cholesterol, triglyceride, homocysteine, orglucose may be placed in the sensor array. Blood serum that is passedover the area may, therefore, be analyzed for glucose, triglyceride, andcholesterol. A key feature of a glucose, triglyceride, homocysteine,and/or cholesterol test is that the test should be able to reveal theconcentration of these compounds in the persons blood. This may beaccomplished using the sensor array by calibrating the reaction of theparticles to cholesterol, triglyceride, or glucose. The intensity of thesignal may be directly correlated to the concentration. In anotherembodiment, multiple particles may be used to detect, for example,glucose. Each of the particles may be configured to produce a signalwhen a specific amount of glucose is present. If the glucose present isbelow a predetermined concentration, the particle may not produce adetectable signal. By visually noting which of the particles areproducing signals and which are not, a semi-quantitative measure of theconcentration of glucose may be determined. A similar methodology may beused for cholesterol, triglyceride, homocysteine, or any testing systemthereof (e.g., glucose/cholesterol/triglyceride/homocysteine,cholesterol/triglyceride, glucose/triglyceride, glucose/cholesterol,etc.).

Another use for the sensor array system is in hormone testing. The mostcommon types of hormone testing in use today are fertility testingdevices (e.g., pregnancy tests and ovulation tests). Both of these teststypically rely on either an immunoassay or enzyme assay methodology.Other hormones, such as progesterone for fertility monitoring orestrogen for hormone therapy treatments may also be monitored. Thesensor array may be used in hormone testing for specific hormones or formultiple hormones in a manner similar to that described forglucose/cholesterol testing.

Another practical use for the sensor array system is for therapeuticdrug monitoring. Therapeutic drug monitoring is the measurement of theserum level of a drug and the coordination of this serum level with aserum therapeutic range. The serum therapeutic range is theconcentration range where the drug has been shown to be efficaciouswithout causing toxic effects in most people. Typically, therapeuticdrug monitoring relies on the analysis of blood serum or plasma from apatient. In general, therapeutic drug monitoring relies on the use ofimmunoassays, similar to the ones described previously.

A general problem with monitoring of drug serum levels may occur when apatient is using more than one drug. In some instances, the drugs mayproduce a positive result in an immunoassay, especially if the drugshave a similar chemical structure. In some instances, the receptor(antibody or antigen) may be altered to prevent a particularinterference. The use of a sensor array, however, may avoid thisproblem. Because a sensor array may include a variety of differentparticles, each of the particles may be customized for a particulardrug. If multiple drugs are present in a patients serum, the presence ofthe drugs may be determined by observing which of the particles isactivated. Even though some of the particles may be reactive to morethan one of the drugs, other receptors may be more finely tuned to aspecific drug. The pattern and intensity of the reactions of theparticles with the drugs may be used to accurately assess the drugspresent in the patient.

One area of therapeutic monitoring includes the monitoring ofanticonvulsant drugs. Anticonvulsant drugs are usually measured by animmunoassay. Common anticonvulsant drugs that require monitoring includephenyloin (Dilantin®), carbamazepine (Tegretol®), valproic acid(Depakene®), primidone (Mysoline®), and phenobarbital. Since primidoneis metabolized to phenobarbital, both drugs must be measured when thepatient is taking primidone.

Another of therapeutic drug monitoring that a sensor array may be usedfor is the monitoring of Digoxin. Digoxin is a medicine that slows theheart and helps it pump more effectively. The bioavailability ofdifferent oral preparations of digoxin tends to be highly variable frompatient to patient. Digoxin measurements may be made using animmunoassay. Some immunoassays for digoxin, however, havecross-reactivity with a hormone-like substance know as digoxin-likeimmunoreactive factor, or DLIF. Care must be taken to distinguishbetween digoxin and digitoxin, another cardiac glycoside. Digoxin assaysgenerally have a low cross-reactivity with digitoxin, but digitoxinserum therapeutic levels may be 10 times those of digoxin. The use of asensor array system that includes a variety of particles, some of whichare more sensitive to DLIF or digitoxin, may allow a more accurateassessment of digoxin levels in a patient.

Theophylline is a bronchodilator with highly variable inter-individualpharmacokinetics. Serum levels must be monitored after achievement ofsteady-state concentrations to insure maximum therapeutic efficacy andprevent toxicity. Immunoassay is the most common method used formonitoring this drug.

Lithium compounds are used to treat bipolar depressive disorders. Serumlithium concentrations are measured by ion selective electrodetechnology. An ion selective electrode has a membrane which allowspassage of the ion of interest but not other ions. A lithium electrodewill respond to lithium concentrations but not to other small cationssuch as potassium. Several small analyzers which measure lithium usingion selective electrode technology are available. The use of particlesthat are sensitive to lithium ion concentrations, as have been describedpreviously, may allow lithium ion measurements to be preformed withoutthe use of lithium ion electrodes. Such systems will allow the analysisof multiple ions in the serum, unlike the electrode based systems whichare specific for lithium ions.

The tricyclic antidepressant drugs include imipramine and itspharmacologically active metabolite desipramine; amitriptyline and itsmetabolite nortriptyline; and doxepin and its metabolite nordoxepin.Both the parent drugs and the metabolites are available aspharmaceuticals. These drugs are primarily used to treat bipolardepressive disorders. Imipramine may also be used to treat enuresis inchildren, and severe Attention Deficit Hyperactivity Disorder that isrefractory to methylphenidate. Potential cardiotoxicity is the majorreason to measure these drugs. Immunoassay methods are available formeasuring imipramine and the other tricyclics. When measuring tricyclicantidepressants which have pharmacologically active metabolites, it isimportant to measure both the parent drug and the metabolite. A sensorarray system is well suited for this type of analysis. Mixtures ofreceptors for parent drug and the metabolites may be incorporated into asingle sensor array. In addition, a sensor array system may be used todetect a variety of tricyclic antidepressant drugs, allowing any of thedrugs to be tested using a single test.

Screening patients for drugs of abuse in the urine may be indicated tohelp differentiate symptoms, or to insure that a patient issubstance-free before undergoing medical procedures. Drug screening ofpregnant women with a history of drug abuse may be useful as aneducational tool and help guide treatment of the newborn. In addition,some employers require a drug screen as part of an employment orpre-employment physical. Nearly all workers in some occupations, such aslaw enforcement and transportation, are subject to periodic, random, andpost-incident drug screening. The chemical sensor array may be used todetect a variety of drugs of abuse in a quick and easy manner.Typically, a variety of different tests must be used to test for eachclass of drug. By incorporating multiple particles into a single sensorarray, some or all of the most commonly used drugs of abuse may bedetermined in a single step.

Urine screening tests for drugs of abuse detect general classes ofcompounds, such as amphetamines, barbiturates, benzodiazepines, oropiates. Drug screening also includes testing for cocaine, marijuana,and phencyclidine (PCP). The screening test for cocaine detects benzoylecgonine, the major metabolite of cocaine. The marijuana test detectsD-9-tetrahydrocannabinol, a principle product of marijuana smoke. Oneproblem of the screening test is that the test, in some instances, maynot be able to distinguish between illicit drugs and prescription orover-the-counter compounds of the same class. A patient taking codeineand another taking heroin would both have a positive screening test foropiates. Some over-the-counter medications can cause a positive drugscreen in a person who has not taken any illegal or prescription drugs.For instance, over-the-counter sympathomimetic amines such aspseudoephedrine and phenylpropanolamine may cause a false-positivescreen for amphetamines. Eating food containing poppyseeds may result ina positive urine screening test for opiates, since poppyseeds containnaturally-occurring opiates. However, confirmation testing willdistinguish between positive opiate tests resulting from poppyseedingestion and those resulting from heroin or other opiates, becausedifferent metabolic breakdown products are present. Monoacetylmorphine(also called 6-monoacetylmorphine or 6-MAM) is a heroin metabolite. Thepresence of this metabolite is conclusive evidence that heroin wasingested.

Most of these problems of false positive results may be avoided throughthe use of a sensor array. The sensor array may include a variety ofparticles, each specific for a particular drug. Some of the particlesmay be specific designed to interact with the drug of abuse, for exampleamphetamine. Other particles may be designed to interact with anover-the-counter drug such as pseudoephedrine. The use of a variety ofparticles may allow a more accurate or complicated analysis to beperformed through the use of a pattern recognition system. Even thoughmany of the drugs may react with one or more particles, the pattern andintensity of the signals produced by the particles in the sensor arraymay be used to determine the identity of the drugs present in thepatient. The most commonly used test method for screening urine fordrugs of abuse is immunoassay. A number of single use devicesincorporating immunoassays and designed to be used outside of thetraditional laboratory are currently available.

Hyperglycemia can be diagnosed only after ruling out spuriousinfluences, especially drugs, including caffeine, corticosteroids,indomethacin, oral contraceptives, lithium, phenyloin, furosemide,thiazides, etc. Thus, a sensor array may be used to expedite diagnosisof hyperglycemia by determining the presence of drugs that may causefalse positives.

In another embodiment, a sensor array may be used to assess the presenceof toxins in a person or animal's system. In general, toxins may be anysubstance that could be ingested that would be detrimental to one'shealth. For animals, a few examples of toxins include lead, organicphosphates, chlorinated hydrocarbons, petroleum distillates, alkaloids(present in many types of poisonous plants), ethylene glycol, etc.People may ingest a variety of these compounds, along with a number ofdifferent types of drugs, either over-the counter, prescription, orillegal. In many instances the patient, either animal or human, mayexhibit symptoms which indicate the presence of a poison, however, thediagnosis of the particular poison ingested by the person may bedifficult. This may be particularly difficult for animals or children,since the owner may not know what the animal/child has eaten. Forpeople, if the poisoning is severe, the person may be unconscious andunable to tell the physician the cause of the poisoning.

The use of a sensor array, may allow a medical expert to accurately andquickly assess the types of toxins present in a patient. A single sensorarray may hold particles that are reactive to a wide variety of toxins.A single analysis of a sample of the patients bodily fluids (e.g.,blood) may allow the medical expert to determine the identity of thepoison. Once identified, the proper treatment may be used to help thepatient.

A sensor array may also be used for soil testing. As with the graintesting, the testing of soil samples may require an extraction of thesoil samples by a suitable solvent. For metals and other inorganicsalts, the solvent used may be either water or dilute aqueous acidsolutions. The soil may also be extracted with organic solvents toextract any organic compounds that are present in the soil sample. Thesesolution containing the extractable material may then be analyzed usinga sensor array. The sensor array may include particles that are specificfor a variety of soil contaminates such as paints, lead, phosphates,pesticides, petroleum products, industrial fallout, heavy metals, etc.The use of a sensor array may allow one or more of these materials to besimultaneously analyzed in a soil sample.

EXAMPLES

In the below recited table are examples of analytes that have beendetected using the sensor array system described herein. In theReceptor/Enzyme column are listed examples of receptors that may be usedfor the corresponding analyte. These receptors are covalently bound to apolymeric resin, using methods described herein.

Analyte Type Receptor/Enzyme Sodium, Potassium Small Molecule(Electrolyte) Crown ethers, cryptands, chromoionophores such asChromolyte ® (from Bayer), Enzymes such as (β- galactosidase, or othermetalloenzymes. Bicarbonate Small Molecule (Electrolyte) Enzymes such asCarbonic anhydrase Calcium Small Molecule (Electrolyte) Complexometricdyes such as Arsenazo III, Xylenol Orange, Alizaren Complexone MagnesiumSmall Molecule (Electrolyte) Complexometric dyes such as Calmagite,Magon Chloride Small Molecule (Electrolyte) Enzymes and/or smallmolecule detectors such as Amylase, Phenyl mercury compounds, mercuricthiocynanates, diphenylcarbazones Oxygen Small Molecule (Metabolite)Oxygen complexing molecules such as porphyins, synthetic hemeglobins,Ruthenium trisbipyridine Carbon dioxide Small Molecule (Metabolite)Enzymes such as Carbonic anhydrase pH Small Molecule (Electrolyte) PHindicator dyes such as Hydroxynitrophenylacetic acid, Congo Red,Brilliant Yellow, Carboxyphenolphthalein Creatinine Small Molecule(Metabolite) Enzymes such as Creatinine deiminase or small moleculedetectors such as picrate Urea Small Molecule (Metabolite) Enzymes suchas Urease Glucose Small Molecule (Metabolite) Enzymes such as Gluocoseoxidase/Peroxidase Hepatitis B Virus Antigen/antiboby pairs such asHepatitis B surface antigen Feline Leukemia Virus Antigen/antiboby pairssuch as FeLV antigen Cytokines Interleukin Small Molecule (Markers),Small molecule markers and/or 1 Interleukin 2 Cellular signalsantigen/antibody pairs Interleukin 4 Interleukin 6 Interleukin 10 GammaInterferon Tumor Necrosis Factor (TNF)

Nucleic Acid Identification Methodology

In one embodiment, the chemical sensor array may be used for thedetermination of the sequence of nucleic acids. Generally, a receptormay be attached to a polymeric bead to form a particle. The receptor mayhave a specificity for a predetermined sequence of a nucleic acid.Examples of receptors include deoxyribonucleic acids (DNA) natural orsynthetic (e.g., oligomeric DNA), ribonucleic acids (RNA) natural orsynthetic, and enzymes. A number of methods may be used to analyze anucleic acid to determine its sequence. The methods, summarized below,may be adapted for use in the previously described chemical sensor arrayto analyze a sample which includes a nucleic acid analyte.

In one embodiment, hybridization may be used to identify nucleic acids.This method relies on the purine-pyrimidine pairing properties of thenucleic acid complementary strands in the DNA-DNA, DNA-RNA and RNA-RNAduplexes. The two strands of DNA are paired by the establishment ofhydrogen bonds between the adenine-thymine (A-T) bases and theguanine-cystosine (G-C) bases. Hydrogen bonds also form theadenine-uracil (A-U) base pairs in the DNA-RNA or RNA-RNA duplexes.Hybridization is highly sequence dependent. Sequences have the greatestaffinity with each other where, for every purine in one sequence(nucleic acid) there exists a corresponding pyrimidine in the othernucleic acid and vice versa. The target fragment with the sequence ofinterest is hybridized, generally under highly stringent conditions thattolerate no mismatches. U.S. Pat. No. 6,013,440 to Lipshutz, et al.describes hybridization in further detail and is incorporated byreference as if fully set forth herein.

Despite the high specificity of hybridization, there may be somemismatched nucleic acid strands. There are several ways to preventmismatched strands from causing false positives. Ribonuclease enzymesmay be used to dispose of mismatched nucleic acid pairs forming aRNA/DNA or RNA/RNA hybrid duplex. There are many types of ribonucleaseenzymes that may be used for this purpose, including RNase A, RNase T1and RNase T2. Ribonuclease enzymes specifically digest single strandedRNA. When RNA is annealed to form double stranded RNA or an RNA/DNAduplex, it may no longer be digested with these enzymes. When a mismatchis present in the double stranded molecule, however, cleavage at thepoint of mismatch may occur. In one embodiment, a label may be attachedto the RNA coupled to the particle. In the presence of a mismatch,cleavage may occur at the point of the mismatch. The cleavage may causethe labeled fragment to fall off the bead, causing a decrease in thesignal detected from the bead. If the nucleic acid are perfectlycomplementary, then the fragment may remain uncleaved in the presence ofthe ribonuclease enzymes and the intensity of the signal produced by theparticle may remain unchanged.

S1 Nuclease Cleavage may also be used to cleave mismatched pairs. S1nuclease, an endonuclease specific for single-stranded nucleic acids,may recognize and cleave limited regions of mismatched base pairs inDNA:DNA or DNA:RNA duplexes. Normally, for S1 Nuclease to recognize andcleave a duplex a mismatch of at least about four consecutive base pairsis required. In a similar manner as described above, the cleavage of alabeled nucleic acid fragment may indicate the presence of a mismatchednucleic acid duplex.

T4 endonuclease VII (T4E7) and T7E1 are small proteins frombacteriophages that bind as homodimers and cleave aberrant DNAstructures including Holliday Junctions. These molecules preferentiallycleave mismatched duplexes. (Described in Youil R, Kemper B, Cotton RGH.Detection of 81 of 81 Known Mouse Beta-Globin Promoter Mutations With T4Endonuclease-VII—The EMC Method. Genomics 1996; 32:431-5, incorporatedby reference as if fully set forth herein).

In another method, Chemical Cleavage of Mismatches (CCM) may be used.This technique relies upon the use of intercalation. Examples ofintercalators include, but are not limited to, the chemicalshydroxylamine and osmium tetroxide to react with a mismatch in a DNAheteroduplex. Mismatched thymines are susceptible to modification byosmium tetroxide (or tetraethyl ammonium acetate and potassiumpermanganate) and mismatched cytosines can be modified by hydroxylamine.The modified bases are then cleaved by hot piperidine treatment. In asimilar manner as described above, the cleavage of a labeled nucleicacid fragment may indicate the presence of a mismatched nucleic acidduplex.

In another embodiment, DNA-binding proteins may be used to identifynucleic acids. Most sequence-specific DNA-binding proteins bind to theDNA double helix by inserting an α-helix into the major groove (Pabo &Sauer 1992 Annu. Rev. Biochem. 61. 1053-1095; Harrison 1991 Nature(London) 353, 715-719; and Klug 1993 Gene 135, 83-92). U.S. Pat. No.5,869,241 to Edwards, et al. describes in detail methods for identifyingproteins having the ability to bind defined nucleic acid sequences andis incorporated by reference as if fully set forth herein. In anembodiment, the DNA-binding proteins may be attached to a polymericparticle. The DNA-binding proteins may interact with the polymericparticle to produce a signal using a variety of the previously describedsignaling protocols.

Mispair Recognition Proteins, e.g., MutS, may also be used to detectmismatched base pairs in double-stranded DNA. There are several methodsby which Mispair Recognition Proteins can be used. Mispair RecognitionProteins may bind to a mismatched base pair. Modified forms of amismatch recognition protein may cleave a heteroduplex in the vicinityof a mismatched pair. A mismatch repair system dependent reaction, e.g.,MutHLS, may be used for mismatch-provoked cleavage at one or more GATCsites. A mismatch repair system may be used in the formation of amismatch-provoked gap in heteroduplex DNA. Mismatch-containingnucleotides may be labeled with a nucleotide analog, e.g., abiotinylated nucleotide. Molecules containing a base pair mismatch maybe removed through the binding of the mismatch to the components of themismatch repair system or by the binding of a complex of a mismatch andcomponents of a mismatch repair system to other cellular proteins.Molecules containing mismatches may also be removed through theincorporation of biotin into such a molecule and subsequent removal bybinding to avidin. The use of Mispair Recognition Proteins is describedin detail in U.S. Pat. No. 6,008,031 to Modrich, et al., which isincorporated by reference as if fully set forth herein. Hsu I C, Yang QP, Kahng M W, Xu J F. Detection of DNA point mutations with DNA mismatchrepair enzymes. Carcinogenesis 1994; 15:1657-62. 1, which isincorporated by reference as if fully set forth herein, describes theuse of MutY in combination with thymine glycosylase for mismatchdetection.

Yet another technique is Oligonucleotide Ligation Assay. In this method,the enzyme DNA ligase is used to join two oligonucleotides, annealed toa strand of DNA, that are exactly juxtaposed. A single base pairmismatch at the junction of the two oligonucleotides will preventligation. Ligation is scored by assaying for labels on the twooligonucleotides becoming present on a single molecule.

In another embodiment, an intercalating molecule may be used as areceptor. The combination of the intercalator with the polymeric beadmay be used as a particle for a sensor array system. Intercalatorstypically react with duplex DNA by insertion into the duplex DNA. If theintercalator has a visible or ultraviolet absorbance or fluorescence,the wavelength or intensity of the intercalators signal may be alteredwhen the intercalator is intercalated into duplex DNA. Examples of suchintercalators include, but are not limited to, ethidium bromide, POTO,and Texas Red. Many intercalators exhibit some sequence selectivity.Thus, an intercalator bound to a polymeric resin may be used toanalyzing DNA analytes for specific sequences. By using a variety ofdifferent intercalators in a single sensor array, the identity of thenucleic acid may be identified through a pattern recognitionmethodology.

The use of particles that are custom made for a variety of differentnucleic acid testing schemes allows greater flexibility than the currentcommercially available nucleic acid devices. For example, the use ofsilicon chips in which the nucleic acid receptor is coupled directly tothe chip may be less flexible since the size of the oligomeric receptorbuilt onto the chip is limited to 25-30 base pairs. Methods forsynthesizing oligomeric nucleic acids on a bead, however, may be used tocouple oligomeric nucleic acids which include more than 100 base pairs.

Tests used to identify nucleic acids sometimes require that the amountof nucleic acid in the sample be increased. Techniques have beendeveloped to amplify the chemical of interest. For example, it ispossible to control which strand of a duplex nucleic acid is amplifiedby using unequal amounts of primer so that the primer for the undesiredstrand is effectively rate limiting during the amplification step.Methods of determining appropriate primer ratios and template sense arewell known to those of skill in the art (see, e.g., PCR Protocols: aGuide to Methods and Applications, Innis et al., eds. Academic Press,Inc. N.Y. 1990).

Polymerase Chain Reaction (PCR) is a widely used technique which enablesa scientist to amplify DNA and RNA sequences at a specific region of agenome by more than a millionfold, provided that at least part of itsnucleotide sequence is already known. The portions on both sides of theregion to be amplified are used to create two synthetic DNAoligonucleotides, one complementary to each strand of the DNA doublehelix, which serve as primers for a series of synthetic reactions whichare catalyzed by a DNA polymerase enzyme. Effective amplification mayrequire up to 30 to 40 repetitive cycles of template nucleic aciddenaturation, primer annealing and extension of the annealed primers bythe action of a thermostable polymerase. A more detailed description aswell as applications of PCR are provided in U.S. Pat. Nos. 4,683,195;4,683,202; and 4,965,188; Saiki et al., 1985, Science 230:1350-1354;Mullis et al., 1986, Cold Springs Harbor Symp. Quant. Biol. 51:263-273;Mullis and Faloona, 1987, Methods Enzymol. 155:335-350; PCRTechnology-principles and applications for DNA amplification, 1989, (ed.H. A. Erlich) Stockton Press, New York; PCR Protocols: A guide tomethods and applications, 1990, (ed. M. A. Innis et al.) Academic Press,San Diego; and PCR Strategies, 1995, (ed. M. A. Innis et al.) AcademicPress, San Diego, Barany, 1991, PCR Methods and Applic. 1:5-16);Gap-LCR(PCT Patent Publication No. WO 90/01069); each of which isincorporated by reference as if fully set forth herein.

In Allele-Specific PCR (also called the amplification refractorymutation system or ARMS) the assay occurs within the PCR reactionitself. Sequence-specific PCR primers which differ from each other attheir terminal 3′ nucleotide are used to only amplify the normal allelein one reaction, and only the mutant allele in another reaction. Whenthe 3′ end of a specific primer is fully matched, amplification occurs.When the 3′ end of a specific primer is mismatched, amplification failsto occur.

Other amplification techniques include Ligase Chain Reaction, describedin Wu and Wallace, 1989, Genomics 4:560-569 and Barany, 1991, Proc.Natl. Acad. Sci. USA 88:189-193, incorporated by reference as if fullyset forth herein; Strand Displacement Amplification; Nucleic AcidSequence Base Amplification; Transcription Mediated Amplification;Repair Chain Reaction, described in European Patent Publication No.439,182 A2), 3SR (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA86:1173-1177; Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA87:1874-1878; PCT Patent Publication No. WO 92/0880A), and NASBA (U.S.Pat. No. 5,130,238), incorporated by reference as if fully set forthherein; Self-sustained Sequence Replication; Strand DisplacementAmplification, etc., described in Manak, DNA Probes, 2^(nd) Edition, p255-291, Stockton Press (1993)), incorporated by reference as if fullyset forth herein; and Non-Isotopic RNase Cleavage Assay, described inGoldrick M M, Kimball G R, Liu Q, Martin L A, Sommer S S, Tseng J Y H.Nirca™—A Rapid Robust Method For Screening For Unknown Point Mutations.Biotechniques 1996; 21:106-12, incorporated by reference as if fully setforth herein. Non-Isotopic RNase Cleavage Assay amplifies RNA. RNaseenzymes, e.g., RNase 1 and RNase T1, increase the sensitivity of theassay.

Manufacturing Methods for a Sensor Array

As described above, after the cavities are formed in the supportingmember, a particle may be positioned at the bottom of a cavity using amicromanipulator. This allows the location of a particular particle tobe precisely controlled during the production of the array. The use of amicromanipulator may, however, be impractical for mass-production ofsensor arrays. A number of methods for inserting particles that may beamenable to an industrial application have been devised.

In one embodiment, the use of a micromanipulator may be automated.Particles may be “picked and placed” using a robotic automated assembly.The robotic assembly may include one or more dispenser heads. Adispenser head may be configured to pick up and hold a particle.Alternatively, a dispenser head may be configured to hold a plurality ofparticles and dispensing only a portion of the held particles. Anadvantage of using a dispense head is that individual particles or smallgroups of particles may be placed at precise locations on the sensorarray. A variety of different types of dispense heads may be used.

In one embodiment, a vacuum pick-up/dispense head may be used. Thedispense head uses a vacuum system to pick up particles. The dispensehead may be formed using small diameter tubing, with an inner diameter(ID) smaller than the particle outer diameter (OD). The dispense headmay be coupled to a robotic control system via an arm. The roboticcontrol system may be programmed to first move the dispense head to astorage location of the correct particle type, vacuum would be appliedto the dispense head once it is “dipped” into the particle storagecompartment, thus grasping one particle. The robotic control systemwould then move the arm such that the dispense head is in a position inclose proximity to (or actual contact with) the appropriate location onthe sensor array (See FIG. 70A). The dispense head vacuum would then beturned off (i.e., the vacuum would be removed), and if necessary aslight positive pressure could be applied to the dispense head. Theparticle would thus be dislodged from the dispense head onto the sensorarray (See FIG. 70B).

The robotic control system may include a single dispense head or aplurality of dispense heads. The use of a plurality of dispense headswould allow multiple cavities of the sensor array to be filled during asingle filing operation. In this manner the efficiency of filling thesensor array may be increased.

Another example of a robotic vacuum pick-up/dispense head is describedin U.S. Pat. No. 6,151,973 to Geysen which is incorporated herein byreference.

The dispense head could also be in the form of a “solid” pick-up wand.The solid dispense head may rely on natural attractive forces between aparticle and the dispense head material to attach a particle to thedispense head. For example, when a particle is placed in close proximityto the dispense head, electrostatic interactions between the particleand the dispense head may cause the particle to “stick” to the dispensehead. The dispense head may be placed at the appropriate location over acavity of the sensor array (See FIG. 71A). When the particle is placedin close proximity to the sensor array, the attractive forces betweenthe chip and particle, along with gravitational forces, may cause theparticle to transfer from the dispense head to the sensor array (SeeFIG. 71B). For example, with PEG particles, a dispense head made oftungsten will cause the PEG particle to attach to the tungsten tip, butthe particle may still be transferred to a silicon based sensor arraywhen brought into close proximity of the sensor array. A single soliddispense head or a plurality of solid dispense heads may be used.

In another embodiment, the dispense head could also be formed from oneor more “pipettes” with an inner diameter greater than the diameter ofthe particles. Particles may be delivered directly into the bore of thepipette using a pump/dispense system. Such a system is similar toprecision adhesive dispense systems in current use. The particles may besuspended in a liquid (e.g., water), and controlled amounts of theliquid would be pumped through the head to deliver a particle to theappropriate location on the sensor array chip. Such a dispensing systemmay have difficulties delivering only one particle at a time. Any extraparticles, however, may be removed form the sensor array afterapplication. Additionally, by making an array of pipettes the rate ofparticle placement may be increased. Other advantages of this approachmay include the ability to deliver the particles in an aqueousenvironment if the particle chemistry so requires, as well making thedeliver of different particles to each head fast and efficient, since no“pick up” step is required.

The “pipette” system relies on the use of controlled amounts of liquidto transport the particle from a storage area to the tip of the dispensehead. In one embodiment, blast of air may be used to force a portion ofthe liquid toward the dispense head tip. In another embodiment, thedispense head may be made using technology essentially identical to thatused in “ink-jet” printer heads. These heads typically rely on bursts ofheat to quickly heat the liquid, causing bubbles of the liquid to beforced to the tip of the dispense head.

Once the pick-up/dispense head has delivered a particle or collection ofparticles to the appropriate location on the sensor array it may bedesirable to insure that a single particle be collected at exactly thecorrect position on the sensor array. This may be accomplished using avacuum chuck-like effect, as illustrated in FIGS. 72A-72D.

In one embodiment, the sensor array includes cavities used to locate andat least partially contain the particles. When placed on a main vacuumchuck, each individual cavity may also acts as a vacuum chuck. Thesensor array, when placed on a vacuum chuck may allow air-flow throughthe cavities. FIG. 72A depicts a multi-tip dispense head that allows thesimultaneous application of many particles. The head is aligned to thecavities in the sensor array using an appropriate mechanical alignmentsystem. If a particle is simply brought into proximity with the cavity,the fluid (e.g., air, but could also be a liquid) flow through thecavity may draw the particle into its proper location and hold it there(as depicted in FIG. 72B). In some embodiments, the dispense head maydelivered more than one particle to a given cavity. Only one of thedispensed particles, however, is fully held in place by the vacuum atany give cavity. After the dispense head is moved away from the sensorarray, excess particles may be removed using a side-directed jet (air orsome other fluid) as depicted in FIG. 72C. The desired particles areheld in their storage pits by the pressure differential across pitsproduced by the vacuum chuck. The process may now be repeated. In FIG.72D another pipette head is illustrated that dispenses a distinct set ofparticles from that dispensed by the first head. This may allow morerapid dispensing of a larger variety of particle types.

When the sensor array is placed on a vacuum chuck, the particles may bepicked up with a vacuum dispense head. The particles may then be pulledoff of the dispense tool when the vacuum of the dispense head isreleased. The applied vacuum from the vacuum chuck may keep theparticles from in the cavities. After the particles have been dispensed,a cover may be disposed on the sensor array to keep the particles inplace. The cover may be attached to the sensor array using a pressuresensitive adhesive. After the cover is placed onto the sensor array, thevacuum may be released and the sensor array removed from the vacuumchuck.

Passive Transport of Fluid Samples

For some chemical sensor array systems, fluids may be transported intoand across the sensor array during use. In one embodiment, fluids my betransferred into and through a sensor array using a system that relieson variations in the surface wetting characteristics of a channel. Anadvantage of such a system is that the system may be “passive” (i.e., noexternal power source or components). Upon the introduction of a sample,the samples may be drawn into the system and distributed to theparticles. This is particularly advantageous for small portable sensorarray systems.

In one embodiment, a chemical sensor array is composed of a number ofsuperimposed layers. FIG. 73 depicts a side-sectional view of the sensorarray system. A support layer 1010 (e.g., a glass layer) is used as thefoundation for the system. A spacer layer 1020 is formed upon thesupport layer. The support layer may be formed of a relatively inertmaterial using standard semiconductor lithographic techniques. In oneembodiment, the support layer may be formed from photoresist (e.g., adry film photoresist). Alternatively, silicon nitride or silicon dioxidemay be used as the spacer layer. The spacer layer may be patterned suchthat the spacer layer supports an outer portion of an overlying sensorlayer 1030. This etching of the spacer layer 1020 may form a channel1022 under the cavities formed in the sensor layer 1030. This channel1022, may allow fluids to pass through the cavities and out of thesensor array system.

The sensor layer 1030 includes a number of cavities 1036 for holding aparticle 1038. The formation of cavities in a sensor layer has beendescribed earlier. In one embodiment, the sensor layer is formed fromsilicon. The silicon sensor layer may be partially etched such that aninlet and channel may be formed in the silicon layer. As depicted inFIG. 73 the outer portion of the sensor layer may be thicker than theinterior portions. The application of a cover layer 1050, may beaccomplished by resting the cover layer on the elevated portions of thesensor layer. This creates a channel 1042 between the cover layer andthe sensor layer.

The etched portion of the sensor layer may be divided into segmentscoupled by a channel. FIG. 74A depicts a top view of the sensor arraysystem and FIG. 74B depicts a bottom view. The first segment 1041 actsas a well or reservoir for the introduction of fluid samples. The secondsegment 1045 may include a number of cavities which include particles.The first segment may be coupled to the second segment by one or morechannels 1043, formed in the sensor array. The channels allow the fluidto flow from the reservoir to the cavities. The cover layer 1050 may bepositioned over the support layer 1030 to form channel 1042. Materialsand methods of for forming the cover layer have been describedpreviously.

Referring back to FIG. 73, the conduction of a fluid through the channelmay be accomplished using a combination of hydrophobic and hydrophilicsurfaces. In one embodiment, a series of hydrophobic segments 1032 areapplied to a surface of channels 1022 and 1042. A layer of a hydrophilicmaterial 1034 may be placed on the opposite surface of the channel, withrespect to the hydrophobic materials. When an aqueous fluid sample isintroduced into the channel, the water is attracted toward thehydrophilic layer while being repelled by the hydrophobic layer. Thisattraction/repulsion creates a current within the channel. Thehydrophilic surfaces may be composed of silicon or hexamethyldisilane.The hydrophobic surfaces may be composed of silicon dioxide, siliconnitride, silicon dinitride, siloxane, or silicon oxynitride.

The system depicted in FIG. 73 may cause a current to flow in adirection from the left side toward the right. Thus the fluid,introduced at inlet 1040, may flow through the channel 1042 in adirection toward the particles. After contacting the particles, thefluid may pass thorough the cavity and into the lower channel 1022. Thehydrophilic and hydrophobic portions of the lower channel may induce acurrent that cause the fluid to flow toward the outlet of the sensorarray system.

Likewise, the system depicted in FIG. 77 may cause current to flow in adirection from the left side to toward the right. Alternatively, thefluid may exit through the top portion of the system through the cover.The fluid may be introduced at inlet 1060 and may flow through channel1062. Fluid may then flow through cavity 1064 past particle 1065. Thefluid may also flow through cavity 1066 past particle 1067. The wall1072 prevents the fluid from flowing past cavity 1066 in the in channel1062. After flowing through the cavities, the fluid flows throughchannel 1068 and then up through cavity 1070. The hydrophilic andhydrophobic portions of the lower channel may induce a current thatcause the fluid to flow toward the outlet 1074 of the sensor arraysystem. In addition, FIG. 73 depicts a bubble-trap 1035 that may consistof a wall in a hydrophobic region.

The sensor array may be formed from a plurality of layers. The layersmay be assembled with dry film materials and ultraviolet curable epoxy.The support layer serves as a base for the system. The support layer maybe formed of a variety of materials including, but not limited to glass,silicon nitride, silicon, silicon dioxide, plastic, and dry filmphotoresist. The support layer is depicted in FIG. 75D.

Onto the support layer is formed a spacer layer. The pattern for anembodiment of the spacer layer is depicted in FIG. 75C. The spacer layermay be placed in the locations that will not be directly under thecavities. The spacer layer may allow a channel to be formed under thesensor array.

The sensor layer is formed upon the spacer layer. A pattern for theetching of the sensor layer is depicted in FIG. 75B. The shaded areas1031 represent the portion of the sensor layer that is etched to athickness that is less than the remaining portion of the sensor layer1033. The sensor layer may be formed from a variety of materials,including silicon, plastic, and dry film photoresist, as has beendescribed before. The sensor layer may be aligned with the support layerto allow a channel to be formed under the cavities. The channel mayallow fluids to pass from the sensor array system.

A cover layer is placed over the sensor layer. The etching of the coverlayer may allow an upper channel 1042 to be formed between the sensorlayer and the cover layer. The cover layer, in one embodiment, includesan opening 1052 that allows a fluid to be passed through the cover layerto the sensor layer. A pattern for the cover layer is depicted in FIG.75A. The opening may be aligned with a reservoir section of the sensorlayer.

In general, the use of a passive fluid transport system allows only asingle use of the sensor array. Although the sensor array may have manychemical particles, and hence has multi-analyte capability, the surfacewetting “pump” may only be used once. For many testing situations (e.g.,medical testing) this is not a significant problem, since it isdesirable to dispose of the sensing element after a single use. Ifmultiple testing of samples is to be performed an “array of arrays” maybe used, as depicted in FIG. 76. In this case, multiple sampleintroduction sites, each coupled to its own suite of sensor sites, maybe fabricated. This setup may allow multiple uses of the sensor array(i.e., use one sensor suite for each test) or allow the simultaneousanalysis of multiple samples.

Portable Sensor Array System

A sensor array system becomes most powerful when the associatedinstrumentation may be delivered and utilized at the application site.That is, rather than remotely collecting the samples and bringing themto a centrally based analysis site, it may be advantageous to be able toconduct the analysis at the testing location. Such a system may be use,for example, for point of care medicine, on site monitoring of processcontrol applications, military intelligence gathering devices,environmental monitoring, and food safety testing.

An embodiment of a portable sensor array system is depicted in FIG. 78.The portable sensor array system would, in one embodiment, have a sizeand weight that would allow the device to be easily carried by a personto a testing site. The portable sensor array system includes a lightsource, a sensor array, and a detector. The sensor array, in someembodiments, is formed of a supporting member which is configured tohold a variety of particles in an ordered array. The particles are, insome embodiments, elements which will create a detectable signal in thepresence of an analyte. The particles may include a receptor moleculecoupled to a polymeric bead. The receptors may be chosen for interactingwith specific analytes. This interaction may take the form of abinding/association of the receptors with the analytes. The supportingmember may be made of any material capable of supporting the particles.The supporting member may include a plurality of cavities. The cavitiesmay be formed such that at least one particle is substantially containedwithin the cavity. The sensor array has been previously described ingreater detail.

The portable sensor array system may be used for a variety of differenttesting. The flexibility of the sensor array system, with respect to thetypes of testing, may be achieved through the use of a sensor arraycartridge. Turning to FIG. 78, a sensor array cartridge 1010 may beinserted into the portable sensor array system 1000 prior to testing.The type of sensor array cartridge used will depend on the type oftesting to be performed. Each cartridge will include a sensor arraywhich includes a plurality of chemically sensitive particles, each ofthe particles including receptors specific for the desired task. Forexample, a sensor array cartridge for use in medical testing fordiabetes may include a number of particles that are sensitive to sugars.A sensor array for use in water testing, however, would includedifferent particles, for example, particles specific for pH and/or metalions.

The sensor array cartridge may be held in place in a manner analogous toa floppy disk of a computer. The sensor array cartridge may be inserteduntil it snaps into a holder disposed within the portable sensor system.The holder may inhibit the cartridge from falling out from the portablesensor system and place the sensor in an appropriate position to receivethe fluid samples. The holder may also align the sensor array cartridgewith the light source and the detector. A release mechanism may beincorporated into the holder that allows the cartridge to be releasedand ejected from the holder. Alternatively, the portable sensor arraysystem may incorporate a mechanical system for automatically receivingand ejecting the cartridge in a manner analogous to a CD-ROM typesystem.

The analysis of simple analyte species like acids/bases, salts, metals,anions, hydrocarbon fuels, solvents may be repeated using highlyreversible receptors. Chemical testing of these species may berepeatedly accomplished with the same sensor array cartridge. In somecases, the cartridge may require a flush with a cleaning solution toremove the traces from a previous test. Thus, replacement of cartridgesfor environmental usage may be required on an occasional basis (e.g.,daily, weekly, or monthly) depending on the analyte and the frequency oftesting

Alternatively, the sensor array may include highly specific receptors.Such receptors are particularly useful for medical testing, and testingfor chemical and biological warfare agents. Once a positive signal isrecorded with these sensor arrays, the sensor array cartridge may needto be replaced immediately. The use of a sensor array cartridge makesthis replacement easy.

Fluid samples may be introduced into the system at ports 1020 and 1022at the top of the unit. Two ports are shown, although more ports may bepresent. One 1022 may be for the introduction of liquids found in theenvironment and some bodily fluids (e.g., water, saliva, urine, etc.).The other port 1020 may be used for the delivery of human whole bloodsamples. The delivery of blood may be accomplished by the use of apinprick to pierce the skin and a capillary tube to collect the bloodsample. The port may be configured to accept either capillary tubes orsyringes that include blood samples.

For the collection of environmental samples, a syringe 1030 may be usedto collect the samples and transfer the samples to the input ports. Theportable sensor array system may include a holder that allows thesyringe to be coupled to the side of the portable sensor array system.One of the ports 1020 may include a standard luer lock adapter (eithermale or female) to allow samples collected by syringe to be directlyintroduced into the portable sensor array system from the syringe.

The input ports may also be used to introduce samples in a continuousmanner. The introduction of samples in a continuous manner may be used,e.g., to evaluate water streams. An external pump may be used tointroduce samples into the portable sensor array system in a continuousmanner. Alternatively, internal pumps disposed within the portablesensor array system may be activated to pull a continuous stream of thefluid sample into the portable sensor array system. The ports are alsoconfigured to allow the introduction of gaseous samples.

In some cases it may be necessary to filter a sample prior to itsintroduction into the portable sensor array system. For example,environmental samples may be filtered to remove solid particles prior totheir introduction into the portable sensor array system. Commerciallyavailable nucleopore filters 1040 anchored at the top of the unit may beused for this purpose. In one embodiment, filters 1040 may have luerlock connections (either male or female) on both sides allowing them tobe connected directly to an input port and a syringe.

In one embodiment, all of the necessary fluids required for thechemical/biochemical analyses are contained within the portable sensorarray system. The fluids may be stored in one or more cartridges 1050.The cartridges 1050 may be removable from the portable sensor arraysystem. Thus, when a cartridge 1050 is emptied of fluid, the cartridgemay be replaced by a new cartridge or removed and refilled with fluid.The cartridges 1050 may also be removed and replaced with cartridgesfilled with different fluids when the sensor array cartridge is changed.Thus, the fluids may be customized for the specific tests being run.Fluid cartridges may be removable or may be formed as an integral partof the reader.

The fluid cartridges 1050 may include a variety of fluids for theanalysis of samples. In one embodiment, each cartridge may include up toabout 5 mL of fluid and be used for about 100 tests before beingdepleted. One or more of the cartridges 1050 may include a cleaningsolution. The cleaning solution may be used to wash and/or recharge thesensor array prior to a new test. In one embodiment, the cleaningsolution may be a buffer solution. Another cartridge 1050 may includevisualization agents. Visualization agents may be used to create adetectable signal from the particles of the sensor array after theparticles interact with the fluid sample. In one embodiment,visualization agents include dyes (visible or fluorescent) or moleculescoupled to a dye, which interact with the particles to create adetectable signal. In an embodiment, a cartridge 1050 may be a vacuumreservoir. The vacuum reservoir may be used to draw fluids into thesensor array cartridge. The vacuum cartridge would act in an analogousmanner to the vacutainer cartridges described previously. In anotherembodiment, a fluid cartridge may be used to collect fluid samples afterthey pass through the sensor array. The collected fluid samples may bedisposed of in an appropriate manner after the testing is completed.

In one embodiment, an alpha-numeric display screen 1014 may be used toprovide information relevant to the chemistry/biochemistry of theenvironment or blood samples. Also included within the portable sensorarray system is a data communication system. Such systems include datacommunication equipment for the transfer of numerical data, video data,and sound data. Transfer may be accomplished using either data or analogstandards. The data may be transmitted using any transmission mediumsuch as electrical wire, infrared, RF and/or fiber optic. In oneembodiment, the data transfer system may include a wireless link (notshown) that may be used to transfer the digital chemistry/biochemistrydata to a closely positioned communications package. In anotherembodiment, the data transfer system may include a floppy disk drive forrecording the data and allowing the data to be transferred to a computersystem. In another embodiment, the data transfer system may include aserial or parallel port connection hardware to allow transfer of data toa computer system.

The portable sensor array system may also include a global positioningsystem (“GPS”). The GPS may be used to track the area that a sample iscollected from. After collecting sample data, the data may be fed to aserver, which compiles the data along with GPS information. Subsequentanalysis of this information may be used to generate achemical/biochemical profile of an area. For example, tests of standingwater sources in a large area may be used to determine the environmentaldistribution of pesticides or industrial pollutants.

Other devices may also be included in the portable sensor array that arespecific for other applications. For example, for medical monitoringdevices including but not limited to EKG monitors, blood pressuredevices, pulse monitors, and temperature monitors.

The detection system may be implemented in a number of different wayssuch that all of the detection components fit within the casing of theportable sensor array system. For the optical detection/imaging device,either CMOS or CCD focal plane arrays may be used. The CMOS detectoroffers some advantages in terms of lower cost and power consumption,while the CCD detector offers the highest possible sensitivity.Depending on the illumination system (see below), either mono-chrome orcolor detectors may be used. A one-to-one transfer lens may be employedto project the image of the bead sensor array onto the focal plane ofthe detector. All fluidic components may be sealed away from contactwith any optical or electronic components. Sealing the fluids away fromthe detectors avoids complications that may arise from contamination orcorrosion in systems that require direct exposure of electroniccomponents to the fluids under test. Other detectors such asphotodiodes, cameras, integrated detectors, photoelectric cells,interferometers, and photomultiplier tubes may be used.

The illumination system for calorimetric detection may be constructed inseveral manners. When using a monochrome focal plane array, amulti-color, but “discrete-wavelength-in-time” illumination system maybe used. The simplest implementation may include several LED's (lightemitting diodes) each operating at a different wavelength. Red, green,yellow, and blue wavelength LEDs are now commercially available for thispurpose. By switching from one LED to the next, and collecting an imageassociated with each, colorimetric data may be collected.

It is also possible to use a color focal plane detector array. A colorfocal plane detector may allow the determination of calorimetricinformation after signal acquisition using image processing methods. Inthis case, a “white light” illuminator is used as the light source.“White light” LEDs may be used as the light source for a color focalplane detector. White light LEDs use a blue LED coated with a phosphorto produce a broad band optical source. The emission spectrum of suchdevices may be suitable for calorimetric data acquisition. A pluralityof LEDs may be used. Alternatively a single LED may be used.

Other light sources that may be useful include electroluminescentsources, fluorescent light sources, incandescent light sources, laserlights sources, laser diodes, arc lamps, and discharge lamps. The systemmay also be configured to use external light source (both natural andunnatural) for illumination.

A lens may be positioned in front of the light source to allow theillumination area of the light source to be expanded. The lens may alsoallow the intensity of light reaching the sensor array to be controlled.For example the illumination of the sensor array may be made moreuniform by the use of a lens. In one example, a single LED light may beused to illuminate the sensor array. Examples of lenses that may be usedin conjunction with an LED include Diffusing plate PN K43-717 Lens JML,PN61874 from Edmund scientific.

In addition to colorimetric signaling, chemical sensitizers may be usedthat produce a fluorescent response. The detection system may still beeither monochrome (for the case where the specific fluorescence spectrumis not of interest, just the presence of a fluorescence signal) orcolor-based (that would allow analysis of the actual fluorescencespectrum). An appropriate excitation notch filter (in one embodiment, along wavelength pass filter) may be placed in front of the detectorarray. The use of a fluorescent detection system may require anultraviolet light source. Short wavelength LEDs (blue to near UV), maybe used as the illumination system for a fluorescent based detectionsystem.

In some embodiments, use of a light source may not be necessary. Theparticles may rely on the use of chemiluminescence, thermoluminescenceor piezoluminescence to provide a signal. In the presence of an analyteof interest, the particle may be activated such that the paticlesproduce light. In the absence of an analyte, the particles may notexhibit produce minimal or no light.

The portable sensor array system may also include an electroniccontroller which controls the operation of the portable sensor arraysystem. The electronic controller may also be capable of analyzing thedata and determining the identity of the analytes present in a sample.While the electronic controller is described herein for use with theportable sensor array system, it should be understood that theelectronic controller may be used with an of the previously describedembodiments of an analyte detection system.

The controller may be configured to control the various operations ofthe portable sensor array. Some of the operations that may be controlledor measured by the controller include: (i) determining the type ofsensor array present in the portable sensor array system; (ii)determining the type of light required for the analysis based on thesensor array; (iii) determining the type of fluids required for theanalysis, based on the sensor array present; (iv) collecting the dataproduced during the analysis of the fluid sample; (v) analyzing the dataproduced during the analysis of the fluid sample; (vi) producing a listof the components present in the inputted fluid sample (vii) monitoringsampling conditions (e.g., temperature, time, density of fluid,turbidity analysis, lipemia, bilirubinemia, etc).

Additionally, the controller may provide system diagnostics andinformation to the operator of the apparatus. The controller may notifythe user when routine maintenance is due or when a system error isdetected. The controller may also manage an interlock system for safetyand energy conservation purposes. For example, the controller mayprevent the lamps from operating when the sensor array cartridge is notpresent.

The controller may also be configured to interact with the operator. Thecontroller preferably includes an input device 1012 and a display screen1014. A number of operations controlled by the controller, as describedabove, may be dependent on the input of the operator. The controller mayprepare a sequence of instructions based on the type of analysis to beperformed. The controller may send messages to the output screen to letthe used know when to introduce samples for the test and when theanalysis is complete. The controller may display the results of anyanalysis performed on the collected data on the output screen.

Many of the testing parameters may be dependent upon the type of sensorarray used and the type of sample being collected. The controller will,in some embodiments, require the identity of the sensor array and testbeing performed in order to set up the appropriate analysis conditions.Information concerning the sample and the sensor array may be collectedin a number of manners. In one embodiment, the sample and sensor arraydata may be directly inputted by the user to the controller.Alternatively, the portable sensor array may include a reading devicewhich determines the type of sensor cartridge being used once thecartridge is inserted. In one embodiment, the reading device may be abar code reader which is configured to read a bar code placed on thesensor array. In this manner the controller can determine the identityof the sensor array without any input from the user. In anotherembodiment, the reading device may be mechanical in nature. Protrusionsor indentation formed on the surface of the sensor array cartridge mayact as a code for a mechanical reading device. The information collectedby the mechanical reading device may be used to identify the sensorarray cartridge.

Other devices may be used to accomplish the same function as the barcode reader. These devices include, but are not limited to, smartcardreaders and RFID systems. The controller may also accept informationfrom the user regarding the type of test being performed. The controllermay compare the type of test being performed with the type of sensorarray present in the portable sensor array system. If an inappropriatesensor array cartridge is present, an error message may be displayed andthe portable sensor array system may be disabled until the propercartridge is inserted. In this manner, incorrect testing resulting fromthe use of the wrong sensor cartridge may be avoided.

The controller may also monitor the sensor array cartridge and determineif the sensor array cartridge is functioning properly. The controllermay run a quick analysis of the sensor array to determine if the sensorarray has been used and if any analytes are still present on the sensorarray. If analytes are detected, the controller may initiate a cleaningsequence, where a cleaning solution is passed over the sensor arrayuntil no more analytes are detected. Alternatively, the controller maysignal the user to replace the cartridge before testing is initiated.

Another embodiment of a portable sensor array system is depicted inFIGS. 79A and 79B. The portable sensor array 1100 includes a body 1110that holds the various components used with the sensor array system. Asensor array, such as the sensor arrays described herein, may be placedin cartridge 1120. Cartridge 1120 may support the sensor array and allowthe proper positioning if the sensor array within the portable sensorsystem. A schematic cross-sectional view of the body of the portablesensor array system is depicted in FIG. 79B. The cartridge 1120, inwhich the sensor array is disposed, extends into the body 1110. Withinthe body, a light source 1130 and a detector 1140 are positionedproximate to the cartridge 1120. When the cartridge 1120 is insertedinto the reader, the cartridge may be held, by the body 110, at aposition proximate to the location of the sensor array within thecartridge. The light source 1130 and detectors 140 may be used analyzesamples disposed within the cartridge. An electronic controller 1150 maybe coupled to detector. The electronic controller 1150 may be configuredto receive data collected by the portable sensor array system.

The electronic controller may also be used to transmit data collected toa computer. An embodiment of a cartridge for use in a sensor arraysystem is depicted in FIG. 80. The cartridge include a carrier body1210, that is formed of a material that is substantially transparent toa wavelength of light used by the detector. IN one embodiment, plasticmaterials may be used. Examples of plastic materials that may be usedinclude polycarbonates and polyacrylates. In one embodiment the body maybe formed from Cyrolon AR2 Abrasion Resistant polycarbonate sheet atthicknesses of 0.118 inches and 0.236 inches. A sensor array gasket 1220may be placed on the carrier body 120. The sensor array gasket 1220, mayhelp reduce or inhibit the amount of fluids leaking from the sensorarray. Leaking fluids may interfere with the testing being performed.

A sensor array 1230 may be placed onto the sensor array gasket. Thesensor array may include one or more cavities, each of which includesone or more particles disposed within the cavities. The particles mayreact with an analyte present in a fluid to produce a detectable signal.Any of the sensor arrays described herein may be used in conjunctionwith the portable reader.

A second gasket 1240, may be positioned on the sensor array. The secondgasket 1240, may be disposed between the sensor array 1230 and a window1250. The second gasket 1240 may form a seal inhibiting leakage of thefluid from the sensor array. The window may be disposed above the gasketto inhibit damage to the sensor array.

The assembly may be completed by coupling a cover 1270 to the body 1210.A rubber spring 1260 may be disposed between the cover and the window toreduce pressure exerted by the cover on the window. The cover may sealthe sensor array, gaskets, and window into the cartridge. The sensorarray, gaskets and window may all be sealed together using a pressuresensitive adhesive. Examples of a pressure sensitive adhesive includeOptimount 237 made by Seal products. Gaskets may be made from polymericmaterials. In one example, Calon II—High Performance material from Arlonmay be used. The rubber spring may be made form a silicon rubbermaterial.

The cover may be removable or sealed. When a removable cover is used thecartridge may be reused by removing the cover and replacing the sensorarray. Alternatively, the cartridge may be a one use cartridge in whichthe sensor array is sealed within the cartridge.

The cartridge may also include a reservoir 1270. The reservoir may beconfigured to hold the analyte containing fluid after the fluids passthrough the sensor array. FIG. 81 depicts a cut away view of thecartridge that shows the positions of channels formed in the cartridge.The channels may allow the fluids to be introduced into the cartridge.The channels also may conduct the fluids from the inlet to the sensorarray and to the reservoir.

In one embodiment, the cartridge body 1210, includes a number ofchannels disposed throughout the body. An inlet port 1282 is configuredto receive a fluid delivery device for the introduction of fluid samplesinto the cartridge. In one embodiment, the inlet port may include a luerlock adapter, configured to couple with a corresponding luer lockadapter on the fluid delivery device. For example, a syringe may be usedas the fluid delivery device. The luer lock fitting on the syringe maybe coupled with a mating luer lock fitting on the inlet port 1282. Luerlock adapters may also be coupled to tubing, so that fluid delivery maybe accomplished by the introduction of fluids through appropriate tubingto the cartridge.

The introduced fluid passes through channel 1284 to channel outlet 1285.Channel outlet 1285 may be coupled to an inlet port on a sensor array(see description of sensor arrays herein). Channel outlet 1285 is alsodepicted on FIG. 80. The fluids travels into the sensor array andthrough the cavities. After passing through the cavities, the fluidexits the sensor array and enters channel 1286 via channel inlet 1287.The fluid passes through channel 1286 to reservoir 1280. To facilitatethe transfer of fluids through the cartridge, the reservoir may includean air outlet port 1288. Air outlet port 1288 may be configured to allowair to pass out of the reservoir, while retaining any fluids disposedwithin the reservoir. In one embodiment, the air outlet port 1288 may bean opening formed in the reservoir that is covered by a semipermeablemembrane. A commercially available air outlet port includes a DURAVENTcontainer vent, available from W. L. Gore. It should be understood,however, that any other material that allows air to pass out of thereservoir, while retaining fluids in the reservoir may be used. Afterextended use the reservoir 1280 may become filled with fluids. An outletchannel 1290 may also be formed extending through the body 1210 to allowremoval of fluids from the body. Fluid cartridges 1292 for introducingadditional fluids into the sensor array may be incorporated into thecartridges.

Magnetic Particle Production and Use

Magnetic particles may be made by different methods. In an embodiment, asolution containing Fe(II) and Fe(III) (typically FeCl₂ and FeCl₃), anda polymer (e.g. a protein) having available coordination sites may betreated (by titration or otherwise) with a strong base such as aqueousammonia in order to precipitate magnetic iron oxides such as magnetite(Fe₃O₄) in a form which is intimately combined with the polymer. Theprecipitation may be typically carried out with rapid stirring andoptional agitation by sonication, in order to produce resuspendablemagnetic-polymer particles.

After precipitation, the particles may be washed and subsequentlyresuspended in a buffer solution at approximately neutral pH. Otherembodiments may involve the use of metals other than iron in thecoprecipitation reaction. In particular, Fe(III) may be replaced by anyof a wide range of transition metal ions. In some cases, iron may becompletely supplanted by appropriately selected transition metal ions.In some cases, the use of metals other than iron produces coloredparticles ranging from white to dark brown.

Magnetic-polymer particles may be produced of varying size. Magneticparticles may be tailor-made to include specific biofunctional ligandsuseful in various analytical, diagnostic, and other biological/medicalapplications. Magnetic particles may be produced with select chemicalreagents that may be useful in various analytical applications.

Subsequent to precipitation and resuspension of the magnetic-polymerparticles, they may be treated with a bifunctional reagent in order tocross-link reactive sites present on the polymer. This cross-linking maybe effective as either an intra-particulate cross-linking in whichreactive sites are bound on the same particle, or may be a reaction ofan extra-particulate ligand which may then be cross-linked to thepolymer on a given particle. In the second case, a bifunctional reagenthaving a relatively short distance between its two functional groupingsmay be desirable to promote linkage between the particle polymer and theextra-particulate species. Conversely, intra-particulate cross-linkingmay be promoted by the use of a bifunctional reagent which may be longerand may not be sterically hindered from bending so that two reactivesites on a single particle may be linked by a single bifunctionalmolecule.

As an alternative to the use of sonication during either theprecipitation or resuspension steps outlined above, another type ofagitation (such as mechanical stirring) may be employed.

Resuspension of the magnetic-polymer particles may be typically carriedout in a low ionic strength buffer system (e.g. 40 mM phosphate). Thebuffer system may enable resuspension of particles which are notresuspendable in non-ionic solutions. In addition to phosphate buffers,borate and sulfate systems may also be used. The association of polymerand metal may result from coordination of metal present duringcoprecipitation by coordination sites on the polymer. It may be thatcertain coordination sites are more “available” than others, based onboth the strength of the coordinate bond which may be formed by theparticular atom, and the spatial hindrances imposed by surroundingatoms. It is known, for instance, that oxygen atoms having a “free”electron pair complex iron more strongly than amine nitrogen atoms and,to an even greater degree, a hydroxyl oxygen atom. Thus, a polymerbearing oxy-acid functional groups may provide better product particlesthan an amine-substituted polymer. Similarly, coordination sites whichmay be freely approached to close distances may yield better performancethan sites which are hindered in either a path of approach or inapproach distance.

The above-described trends may be qualitatively observable in variousexperiments. The presence of “available coordination sites” appearsnecessary to the production of the resuspendable magnetic-polymerparticles. For example, such diverse polymers as natural proteins,synthetic proteins, poly-amino acids, carboxy-poly-alkyls,alkoxy-poly-alkyls, amino-poly-alkyls, hydroxy-poly-alkyls, and variouscopolymers of these have all been demonstrated to produce suitableparticles. In addition, other polymers such as sulfoxy-poly-alkyls,poly-acrylamines, poly-acrylic acid, and substituted poly-alkylenes mayproduce similar particles.

In selecting the transition metals to be employed in the coprecipitationreaction, several criteria may be important. First, the final compoundmust have one or more unpaired electrons in its structure. Second, oneof the metals must possess an available coordination site for bonding toa polymer. Third, the coprecipitate must be capable of forming a cubicclose-packed or hexagonal close-packed (eg. for cubic: spinel or inversespinel) crystalline structure. This last requirement may be due to theneed for a very close packing in order for a compound to be magnetic.

In an embodiment, polymers useful in preparing the magnetic particlesmay be “tailor-made” to include monomers which may exhibit a specificbiofunctional activity. Using such a polymer may permit directprecipitation of a biofunctional magnetic-polymer particle which mayrequire little or no further treatment in order to be useful in assayswhich rely on the particular biofunctional activity of the polymer.

In some embodiments, larger, less stable particles may be useful. Theparticles may be made to agglomerate while still retaining both theirbiofunctional and magnetic characteristics. Agglomeration of theparticles may be accomplished by treatment of a suspension with apredetermined amount of, for example, barium chloride solution. Thistreatment may be designed to cause the particles to settle out ofsuspension in a predetermined period of time in order to allow theperformance of further procedures, or to allow the larger particles tobe easily attracted by relatively small magnets. U.S. Pat. No. 4,795,698to Owen et al., which is incorporated herein by reference, providesfurther details for producing magnetic particles.

Magnetic particles may also be produced from metallocenes and metalhydroxide compounds. These particles may then be incorporated intopolymeric materials to produce magnetically active particles.

Metallocenes are cyclopentadienyl coordinate complexes of metals. Thecyclopentadienyl group, C₅H₅, has long been known to form complexes withmetals or metalloidal atoms. In an embodiment, metallocenes may becyclopentadienyl complexes of transition metals. The transition-metalsmay include, for example, iron (Fe), magnesium (Mg), manganese (Mn),cobalt (Co), nickel (Ni), zinc (Zn) and copper (Cu). Particularly usefulmetallocenes may be ferrocene (C₅H₅)₂Fe, nickelocene, (C₅H₅)₂Ni, andcobaltocene, (C₅H₅)₂Co. Metallocenes have the general formula (C₅H₅)₂M,wherein M is the metal and have a “sandwich” configuration. Thestructure of metallocenes endows these molecules with high thermalstability (e.g., up to about 500° C. for ferrocene).

In an embodiment, an aqueous slurry of the metallocene may be produced.The slurry may be prepared, for example, by combining the metallocenecompound and water, and mixing or by milling in a high energy mill, suchas a sand mill or a ball mill. The length of time for which the slurriesare milled will depend upon the particle size of the product which maybe desired. The slurry may generally contain from about 0.1 to about 40percent (%) by weight of the metallocene. A slurry containing from about20 to about 25% by weight metallocene may be particularly useful.

The aqueous metallocene slurry may be combined with a second aqueousslurry of a metal hydroxide. The choice of metal hydroxide may dependupon the properties of the particles which may be desired. For example,to produce magnetite particles, iron (II) hydroxide (ferrous hydroxide)may be used. Other metal hydroxides which may be used to producemagnetic particles may include cobalt (II) hydroxide, cobalt (III)hydroxide, iron (III) hydroxide and nickel hydroxide. Slurries of thesemetal hydroxides may be prepared by precipitating a salt of the metal(e.g. chloride or sulfate salt) in an aqueous medium using a base, suchas sodium hydroxide or ammonium hydroxide. An aqueous iron (II)hydroxide slurry may be prepared by precipitating an aqueous solution offerrous chloride or ferrous sulfate with ammonium or sodium hydroxide toform ferrous hydroxide (FeO(OH)). The resulting gelatinous precipitateof iron (II) hydroxide may be filtered, and the solid material may becollected, combined with water and milled in a high energy mill to formthe slurry. The metal hydroxide slurry may contain from about 0.1 toabout 40 percent (%) by weight of the metal hydroxide.

The two slurries may be combined and the mixture may be milled in a highenergy mill, such as a commercial ball or sand mill, for a period oftime sufficient to form fine magnetic particles, generally for about 1hour to about 60 hours. Generally, the longer the milling step, thesmaller the particles which may be formed.

In an embodiment, magnetite particles may be formed from iron (II)hydroxide and ferrocene according to the following equation:

2Fe0(OH)+Fe(C₅H₅)₂→Fe₃O₄+2(C₅H₅)+H₂O+H₂ (gas)

The iron (II) hydroxide powder may be milled in intimate contact withthe ferrocene. Over a period of about 20 to 40 hours, the two materialsmay react by slow dissociation of the hydroxide to form magnetite, freecyclopentene, water and hydrogen. It may be necessary to allowsufficient void space in the mill, or to vent the mill periodically toaccommodate the release of the hydrogen gas formed during the reaction.The particles may then be isolated and incorporated into polymericmaterials to produce beads comprising magnetic particles. Additionalproduction details may be found in U.S. Pat. No. 5,071,076 to Chagnon etal., which is incorporated herein by reference.

In an embodiment, colloidal polymer or protein magnetite may be preparedwith highly controllable, polymer/protein magnetite ratios. Typically,the particles may be precipitated from solutions of hydrated ferric andferrous chlorides at 3.5 and 1.5 mg/ml, respectively, with proteincontent ranging from 500 ug/ml to 1.5 mg/ml. After appropriate washing,resuspension and sonication of such precipitates, colloidal,magnetically responsive particles may be produced, wherein the meandiameter of particles may be approximately inversely proportional tostarting protein concentrations. Particles about 20 nanometers or lessin diameter may be obtained at the higher protein concentrations,whereas particles approximately 100 nanometers in diameter may beobtained at the lower end of the range of protein concentrations. It hasbeen found that the ease with which various of these colloidal solutionsmay be salted out may be inversely related to the protein concentrationof the solution and may be directly related to particle size. In otherwords, the smaller, higher protein containing particles may be moredifficult to salt out. These results suggest that the particles havinghigher protein concentration may be more lyophilic, which might beexpected because of the greater interaction between solvent water andprotein, as compared with magnetite. Other possible explanations forthis observed phenomenon may be that the magnetic cores of the largercolloidal particles may be easier to flocculate because of theirmagnetic moments, or that the smaller particles offer relatively largersurface area and consequently more surface charge to be neutralized.

In an embodiment, colloidal, magnetically responsive particles bearing(i) a biospecific binding material having binding affinity for thetarget substance of interest or (ii) a suitable retrieval agent, forexample, anti-fluorescein, where a fluoresceinated receptor for thetarget substance may be used, may be incubated with an appropriatelylabeled specific binding substance and test sample suspected ofcontaining the target substance, under conditions such thatagglomeration of such particles may not occur. Agglomeration may notoccur, for instance, because the binding capacity of the specificbinding substance or the concentration of the target substance in thetest medium may be too low. Following the binding of sufficient labeledsubstance (or inhibition thereof), an agglomerating agent, which may beeither non-specific, or specific, preferably the former, e.g., a simplesalt solution, may be added to the incubation mixture to causeagglomeration. Agglomeration may be brought about by the addition of asecond non-specific agglomerating agent, e.g., an appropriately chosencolloid, if desired.

Alternatively, agglomeration may be effected by means of a specificagglomerating agent capable of cross-linking a component of thecolloidal magnetic particles, such as a specific antibody. The resultingagglomerate may be removed from solution via centrifugation, filtrationor, via magnetic separation. It may also be possible to use anon-specific and/or specific agglomerating agents in variouscombinations, if desired. Thus, second colloid addition plus salting outmay be feasible, as may the use of a second magnetically responsivecolloidal particle bearing a receptor capable of cross-linking with asubstance present on the colloidal protein magnetite initially added tothe test sample.

Another useful application of the conversion of colloidal material to amagnetically separable form by the addition of a second colloid, may beto use protein colloidal magnetite as the agglomerating agent for someother non-magnetic colloidal material, where the latter bears the targetsubstance of interest.

Colloidal reagents and non-specific or specific agglomerating agents maybe added to the test medium simultaneously, rather than sequentially, aspreviously described. This may be accomplished by adding a suitableagglomerating agent to one of the colloidal reagents used in the assay,so that conversion of the colloid takes place after a substantial levelof ligand/receptor interaction has occurred. Further information onproduction of magnetic colloidal particles may be found in U.S. Pat. No.5,108,933 to Liberti et al., which is incorporated herein by reference.

In an embodiment, permanently magnetized materials may be used toproduce magnetic particles. Previously discussed agglomerationtechniques may be used to form particles in which the particlecomposition may encapsulate the magnetic material. In an embodiment, themagnetic material may be suspended in a solution from which theparticles may be formed. As the particles begin to form, due toagglomeration or other methods, the suspended magnetic material may beencapsulated thereby forming a magnetic particle. Magnetic material mayalso be incorporated into particles by physical means. In an embodiment,magnetic materials may be intermixed with particles using methods suchas, but not limited to ball mills, low intensity mixers, and pug mills.A wide variety of magnetized materials may be used in the magneticparticles. Examples of magnetized materials, besides those materialspreviously discussed, may include, but are not limited to materials suchas alnico, ferrite, barium ferrite, strontium ferrite, neodymium ironboron, samarium cobalt, iron oxide, or other ferromagnetic materials.

Upon formation of the magnetic particle, the magnetic particle may befurther modified with target analyte materials. Eventually, the magneticparticles may be placed within the sensor array. In an embodiment, themagnetic particles may be located within the cavity or cavities of asensor array by placement of permanent magnets in such a manner that themagnetic particle may be directed to a particular location, in thisinstance, a cavity in the sensor array. In an embodiment, a permanentmagnet may be located under a cavity of interest. A solution containingsuspended magnetic particles may be allowed to flow over the cavity,wherein a magnetic particle may be directed into the cavity by theinteraction of the magnetic particle and the permanent magnet. Dependingupon the cavity size, other particles may or may not be directed intothe cavity. For example, a cavity only large enough to include onemagnetic particle, may capture one particle, but, based upon spacelimitation, no further particles may be directed into the cavity.Conversely, a cavity large enough to include several particles may haveseveral particles directed toward it before the cavity may no longercapture particles. When the desired cavity or cavities may be filled, acover layer may be added to the substrate to retain the particles asdiscussed in previous sections. Directing magnetic particles to magnetsfor collection or to a particular location are further discussed in U.S.Pat. No. 4,813,277 to Miller, et al, which is incorporated herein byreference.

Permanent magnets may be used to direct magnetic particles intocavities, but other embodiments may be possible. In an embodiment,electromagnets may be located at a desired cavity, such that themagnetic particle may be drawn into the desired cavity. For example, aflow of magnetic particles may be allowed to pass over the sensor array.An electromagnet may be located under a cavity such that as energy maybe supplied to the electromagnet, a flowing magnetic particle may bedirected into the desired cavity. A plurality of cavities may be locatedon the sensor array and a discrete electromagnet may be assigned to eachcavity. Current flow to each electromagnet may be monitored such that amagnetic particle or particles may be directed to individual cavities.By controlling the electrical current to the electromagnets, somecavities may be filled with magnetic particles while other cavities mayremain empty. A second flow of different magnetic particles may beallowed to flow over the sensor array, at which time otherelectromagnets may be activated thereby causing the different magneticparticles to be directed into the currently empty cavities. Thisprocedure may continue using other different magnetic particles untilthe selected cavities may be filled. In this way, various cavities maybe filled with different magnetic particles. Other embodiments may allowlocation of multiple magnetic particles within the same cavity therebyproviding the ability to detect multiple analytes from the same cavity.Other variations of cavities and particles may be possible wherein thevariations may not be limited by the foregoing embodiments. U.S. Pat.No. 5,981,297 to Baselt, which is incorporated herein by reference,further describes the recognition of magnetic particles with magnets

Formation of Cavities With Retaining Projections

In an embodiment, a mask may be deposited on a bulk crystalline (100)silicon substrate to form an integrated cover layer. The cover layer maybe, but is not limited to, silicon nitride, a plastic, silicon dioxide,or a dry film photoresist material. The cover layer may be formed oretched in such a way that, after etching of the silicon substrate,various flexible micromachined projections may be present in the coverlayer. Many types of structures formed on the cover layer may providefor development of flexible projections after etching. Examples ofstructures that may be formed on the cover layer may be, but are notlimited to: star; cross; circle; square; or any other type of formationthat provides for flexible projections after etching. In an embodiment,a cross, formed by an equal length upright with a transverse beam may beformed in the cover layer. An amount of the cover layer may be removedsuch that the substrate may be exposed to an etchant material. Afterremoving the desired amount of cover layer, the substrate may be etchedanisotropically. The etchant may continue to remove the siliconsubstrate until the bounding (111) planes may be reached. The resultingcavity may be a pyramidal shape into the silicon substrate. Thepyramidal shaped cavity may enhance fluid flow. The cavity may be formedsuch that a bottom opening may also be present.

The flexible projections formed from the undercutting of the siliconsubstrate beneath the cover layer may provide a method of retention ofthe particle. In an embodiment, the flexible projections may be producedfrom a mask opening which may be smaller than the underlying cavity. Theparticle may then be manipulated past the flexible projections into thecavity. As the particle passes the flexible projections, the flexibleprojections may be displaced downward until the particle passescompletely by the flexible projections and into the cavity. As theparticle passes the flexible projections, the flexible projections mayreturn to their original position, thereby providing retention of theparticle in the cavity. Retention of the particle in the cavity may bemaintained by the flexible projections during subsequent handling of thesensor array.

FIG. 82 shows the placement of a particle (1) into a cavity. Theparticle may be placed proximate to the cavity on top of the flexibleprojections (2) as shown in (a), at which time a micromanipulator may beused to press the particle past the flexible projections. The flexibleprojections may bend as the particle may be pressed past theprojections, as shown in (b) and (c), until the particle may be placedwithin the cavity. The flexible projections may return to their normalposition, as shown in (d), as the particle moves past the flexibleprojections and is substantially retained within the cavity.

The flexible projections may provide for specific size selection ofparticles to be placed into the cavity. In an embodiment, it may beassumed the particles may have a gaussian distribution. In anon-limiting example, an opening may be provided in the cover layer bythe flexible projections which may be an opening larger than the meansize of the particle times a sigma value. The sigma value as definedhereinafter is the variability in size of a particle around a meanparticle diameter of a gaussian distribution of particles. The bottomopening of the cavity may be an opening smaller than the mean size ofthe particle times a sigma value. If a 10% sigma value of the particlediameters may be assumed and a 10% sigma value of the top and bottomcavity openings, only the next size up or down may have a chance offilling the cavity. Assuming these variables, the probability forplacing a particle the next size up in the cavity may be about one partin one thousand. The probability of placing a particle the next sizedown in the cavity may be about 1 in 300. Reduction in the variabilityof the particle size and reduction in the variability in the top andbottom openings of the cavity may result in a higher percentage ofcorrectly sized particles being placed in the cavity.

In an embodiment, another strategy which may be employed with beadcapture selectivity probability may be the use of three cavities of adesired size to provide triple redundancy. In this strategy, threecavities of the selected size may be used and selection criteriadesigned such that if two cavities contain the correct particle size,the cavities may be considered correctly filled. An error may result iftwo-same sized cavities may be incorrectly filled. The foregoingcriteria may provide for a selection of the probability of placing a toolarge particle of about 1 in 10⁶ and placement of a too small particleof about 1 in 77,000. Error rates may be further reduced by decreasingthe variability of the particle diameter and variability in the cavitytop and bottom openings.

In an embodiment, a particle may be placed in a cavity using varioustechniques such as individual placement of particles. Micromanipulatorsmay be used in the individual placement of a particle. A vacuum or flowsystem may be used to provide for more rapid production of cavity arrayscompared to individual placement of particles into cavities. In anembodiment, a wafer may be fabricated with the correct top and bottomcavity openings to select the desired particle size. A solution ofparticles with a wide range of size distribution may be produced. Thewafer may then be dipped into the solution whereupon a vacuum or flowmay pull the particles past the cavity top flexible projections. A toolarge particle may not pass the top opening and a too small particle maypass through the cavity and out the cavity bottom. A correctly sizedparticle may pass the top opening flexible projections and be retainedon the cavity bottom. In the embodiment, the flexible projections may beused as cavity opening discriminators. Flexing of the projections as theparticle passes may not be necessary.

A combination of correctly sized flexible projections and particles maybe used to produce a backflow preventer and pump. In an embodiment, acavity may be fabricated such that the slits in the cover layer producea rectangular bottom opening. The top layer may be fabricated such thata round opening slightly smaller than the particle may be produced. Theflexible projections of the bottom opening may be designed for placementof a particle into the cavity. The fluid flow may be inhibited orstopped if the flow direction forces the particle against the round topopening. If the flow is reversed, the particle may be forced against theflexible projections and depending upon the design of the flexibleprojections, flow may occur or may be significantly inhibited. Forexample, the flexible projections may be designed such that the slitsmay be as small as possible resulting in a significant decrease inback-flow capabilities. The effect of this embodiment may produce avalve with a high flow coefficient for flow in one direction and a lowflow coefficient in the opposite direction.

The flexible projections may be designed to bend in one direction morefavorably than in the opposite direction. In an embodiment, multiplelithography or deposition steps for producing the cover layer mayprovide a flexible projection which may flex preferably in the directionto allow placement of a particle within the cavity. The flexibility maybe reduced in the direction in which the projections may be required toflex for removal of the particle. Providing enhanced flexibility in onlyone flexural direction may allow reduction of slit size in the coverlayer needed to provide etch access to the silicon substrate.

In an embodiment, the flexible projections may be produced byundercutting the silicon substrate as described previously. The topcover flexible projections and bottom cover opening may be fabricated tothe diameter desired, such that a particle may only be accepted in ashrunken state. The particle to be placed within the cavity may beexposed to a medium in which the particle may be caused to shrink. Theshrunken particle may then be placed within the cavity at which pointthe particle may be exposed to a medium which causes the particle toreturn to it's normal diameter state. The particle may then be capturedwithin the cavity. Correctly designing the swollen state of the particleand the flexible projections, the particle may be retained within thecavity subsequent to further processing of the array.

The sensor array may be used as a method for sorting various sizedparticles. In an embodiment, the sensor array may be fabricated withvarious sized cavities which may capture various sized particles.Depending upon etch time, the cavity sizes may be configured todifferent sizes. A shorter etch time may produce a smaller cavity sizebased upon the depth of the cavity into the substrate.

In an embodiment to provide selection of only one particle size from adistribution of particle sizes, a solution of particles with a widerange of particle size distribution may be allowed to flow over thesubstrate. A vacuum or flow may be used to pull the particles past thecavities etched into the substrate support. Those particles with a toolarge diameter may not be captured by a cavity where the top opening maybe smaller than the particle diameter. The too large particle maycontinue to flow across the sensor array. Those particles with a smallerdiameter than the bottom opening may be drawn into the cavity as theypass the top opening, but the small diameter particle may pass throughthe bottom opening and out of the substrate support. Particle sizessmaller than the top opening, but larger than the bottom opening may bedrawn into the cavity and retained within the cavity. Those particleslarger than the top opening and smaller than the bottom opening may notbe retained on the substrate support. The non-retained particles mayflow away from the substrate support. The flow may then be stopped andthe substrate along with the captured particles may be removed from theflow. A reverse flow may then be used to dislodge the particles into adesired location.

In an embodiment, the array may provide an ability to pick and placemany particles at once. The substrate may be fabricated with top andbottom openings designed to select a certain desired particle size. Asolution of particles may be flowed over the substrate. Those particlesof the desired particle size may be captured by the cavities asdiscussed in the previous section. The flow may then be stopped and thesubstrate, along with the captured particles, may be removed from theflow. A reverse flow may then be used to dislodge the particles into adesired location.

Further modifications and alternative embodiments of various aspects ofthe invention will be apparent to those skilled in the art in view ofthis description. Accordingly, this description is to be construed asillustrative only and is for the purpose of teaching those skilled inthe art the general manner of carrying out the invention. It is to beunderstood that the forms of the invention shown and described hereinare to be taken as the presently preferred embodiments. Elements andmaterials may be substituted for those illustrated and described herein,parts and processes may be reversed, and certain features of theinvention may be utilized independently, all as would be apparent to oneskilled in the art after having the benefit of this description of theinvention. Changes may be made in the elements described herein withoutdeparting from the spirit and scope of the invention as described in thefollowing claims.

1.-99. (canceled)
 100. A sensor array for detecting an analyte in afluid comprising: (a) a supporting member; wherein at least one cavityis formed within the supporting member; (b) a particle positioned withinthe cavity, wherein the particle is configured to produce a signal whenthe particle interacts with the analyte.
 101. The sensor array of claim100, further comprising a plurality of particles positioned within thecavity.
 102. The sensor array of claim 100, wherein the particlecomprises a receptor molecule coupled to a polymeric resin.
 103. Thesensor array of claim 100, wherein the particle has a size ranging fromabout 0.05 micron to about 500 microns in diameter.
 104. The sensorarray of claim 100, wherein the particle has a size ranging from about0.05 micron to about 500 microns in diameter, and wherein the cavity isconfigured to substantially contain the particle.
 105. The sensor arrayof claim 100, wherein the supporting member comprises a plasticmaterial.
 106. The sensor array of claim 100, wherein the supportingmember comprises a silicon wafer.
 107. The sensor array of claim 100,wherein the cavity extends through the supporting member.
 108. Thesensor array of claim 100, wherein the supporting member comprises asilicon wafer, and wherein the cavity is substantially pyramidal inshape and wherein the sidewalls of the cavity are substantially taperedat an angle of between about 50 to about 60 degrees.
 109. The sensorarray of claim 100, wherein the supporting member comprises a siliconwafer, and further comprising a substantially transparent layerpositioned on a bottom surface of the silicon wafer. 110.-111.(canceled)
 112. The sensor array of claim 100, wherein the supportingmember comprises a silicon wafer, and wherein the silicon wafer has anarea of about 1 cm² to about 100 cm².
 113. The sensor array of claim100, further comprising a plurality of cavities formed in the siliconwafer, and wherein from about 10 to about 10 cavities are formed in thesilicon wafer.
 114. The sensor array of claim 100, further comprisingchannels in the supporting member, wherein the channels are configuredto allow the fluid to flow through the channels into and away from thecavity.
 115. (canceled)
 116. The sensor array of claim 100, furthercomprising a detector coupled to the bottom surface of the supportingmember, wherein the detector is positioned below the cavity.
 117. Thesensor array of claim 100, further comprising a detector coupled to thebottom surface of the supporting member, wherein the detector ispositioned below the cavity, and wherein the detector is a semiconductorbased photodetector or a Fabry-Perot type detector.
 118. (canceled) 119.The sensor array of claim 100, further comprising a detector coupled tothe bottom surface of the supporting member, wherein the detector ispositioned below the cavity, and further comprising an optical fibercoupled to the detector, wherein the optical fiber is configured totransmit optical data to a microprocessor.
 120. (canceled)
 121. Thesensor array of claim 100, further comprising a barrier layer positionedover the cavity, the barrier layer being configured to inhibitdislodgment of the particle during use. 122-124. (canceled)
 125. Thesensor array of claim 100, wherein the system comprises a plurality ofparticles positioned within a plurality of cavities, and wherein theplurality of particles produce a detectable pattern in the presence ofthe analyte.
 126. The sensor array of claim 100, further comprisingchannels in the supporting member, wherein the channels are configuredto allow the fluid to flow through the channels into and away from thecavities, and wherein the barrier layer comprises a cover platepositioned upon an upper surface of the supporting member, and whereinthe cover plate inhibits passage of the fluid into the cavities suchthat the fluid enters the cavities via the channels. 127-134. (canceled)135. The sensor array of claim 100, wherein an inner surface of thecavity is coated with a reflective material. 136-340. (canceled)